Methods and compositions for in vivo gene editing based cell-type-specific cellular engineering

ABSTRACT

Disclosed herein include methods and compositions for incorporating an effector gene into the genome of a cell. The method can comprise introducing into a cell a donor nucleic acid comprising a recognition site, a splice acceptor site, a self-cleaving peptide sequence, an effector gene, and an optional transcript stabilization element. The donor nucleic acid can be incorporated into the intron of a target gene differentially expressed in a unique cell type and/or in a cell during a unique cell state via non-homologous end joining (NHEJ)-dependent DNA repair. There are also provided, in some embodiments, methods and compositions for treating a disease or disorder in a subject.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/832,504, filed Apr. 11, 2019; and U.S.Provisional Application No. 62/832,759, filed Apr. 11, 2019. The entirecontents of these applications are hereby expressly incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled30KJ_302421_US, created Apr. 9, 2020, which is 20.0 kilobytes in size.The information in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND Field

The present disclosure relates generally to the field of cellularengineering.

Description of the Related Art

Emerging cellular profiling technologies have revealed a rich diversityof molecularly defined cell types in multiple organs including blood,immune system and the brain. As most disease conditions only affect oneor just a few cell types in the body, precision therapies functionallycorrecting a particular cell type have become a highly attractive avenuefor therapeutic intervention. Technologies that enable to selectivelytarget and control specific cell types and cell states are thereforehighly desirable.

Reliable and selective cell type specific engineering has to date beenachievable only in animals where creating transgenic organisms isethically permissible. Cell types and states here are defined by virtueof their gene expression. In such organisms, cell type specificmanipulation has been achieved by expressing effector genes that bringabout desired functional changes in the cell under the control of genomeintegrated DNA regions. This approach is usually a labor intensive andtime consuming process.

Technical efforts in gaining cell-type-specific functional control incase germline modification of the genome is not feasible, fall into twomajor categories. The first relies on the use of promoter driverconstructs, where the expression of the effector gene is under thecontrol of a cell-type-specifically active gene promoter. Theseconstructs are delivered by viral or non-viral delivery routes. Due tostrict size limitations of the viral delivery route and the fact thatmany gene expression regulatory elements span large DNA regions, only avery limited set of cells in any given organism are reliably targetablethrough this method. An alternative to this approach is to deliverubiquitously expressed effector genes by delivery methods thatfacilitate uptake only in desired cells. Using viral vectors, where thecapsid composition can bias uptake is an example of this strategy. Thisapproach, however, is suitable for targeting developmentally verydisparate cell types where the membrane biophysics is profoundlydistinct and is not suitable for reliable differential targeting of mostcell types in the organism. In summary, none of the currently availabletechnologies provide simple and selective functional control over themajority of distinct cell types in the body.

There is a need in the field for a technology that allows to use activegenetic programs in the cell for selective functional manipulation ofcell types and cell states.

SUMMARY

Disclosed herein include methods of incorporating an effector gene intothe genome of a cell. In some embodiments, the method comprises:introducing into a cell: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and (iii) a donor nucleicacid or a nucleic acid encoding the donor nucleic acid, wherein thedonor nucleic acid comprises a recognition site, a splice acceptor site,a self-cleaving peptide sequence, an effector gene, and an optionaltranscript stabilization element, wherein the cell comprises a targetgene differentially expressed in a unique cell type and/or in a cellduring a unique cell state, wherein the target gene comprises an introncomprising the recognition site, and wherein the targeting molecule iscomplementary to the recognition site and the programmable nuclease iscapable of cleaving the recognition site, whereby the donor nucleic acidis capable of being incorporated into the intron through non-homologousend joining (NHEJ)-dependent DNA repair.

Disclosed herein include methods of incorporating an effector gene intothe genome of a cell. In some embodiments, the method comprises:introducing into a cell: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease, wherein the programmable nucleasecomprises a zinc finger nuclease (ZFN) and/or transcriptionactivator-like effector nuclease (TALEN); and (ii) a donor nucleic acidor a nucleic acid encoding the donor nucleic acid, wherein the donornucleic acid comprises a recognition site, a splice acceptor site, aself-cleaving peptide sequence, an effector gene, and an optionaltranscript stabilization element, wherein the cell comprises a targetgene differentially expressed in a unique cell type and/or in a cellduring a unique cell state, wherein the target gene comprises an introncomprising the recognition site, and wherein the programmable nucleaseis capable of cleaving the recognition site, whereby the donor nucleicacid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair.

Disclosed herein include methods of treating a disease or disorder in asubject. In some embodiments, the method comprises: introducing into acell of a subject in need thereof: (i) a programmable nuclease or anucleic acid encoding the programmable nuclease; (ii) a targetingmolecule or a nucleic acid encoding the targeting molecule, and (iii) adonor nucleic acid or a nucleic acid encoding the donor nucleic acid,wherein the donor nucleic acid comprises a recognition site, a spliceacceptor site, a self-cleaving peptide sequence, an effector gene, andan optional transcript stabilization element, wherein the cell comprisesa target gene differentially expressed in a unique cell type and/or in acell during a unique cell state, wherein the target gene comprises anintron comprising the recognition site, and wherein the targetingmolecule is complementary to the recognition site and programmablenuclease is capable of cleaving the recognition site, whereby the donornucleic acid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair, and thereby theintroducing treats the disease or disorder in the subject.

Disclosed herein include methods of treating a disease or disorder in asubject. In some embodiments, the method comprises: introducing into acell of a subject in need thereof: (i) a programmable nuclease or anucleic acid encoding the programmable nuclease, wherein theprogrammable nuclease comprises a zinc finger nuclease (ZFN) and/ortranscription activator-like effector nuclease (TALEN); and (ii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid, whereinthe donor nucleic acid comprises a recognition site, a splice acceptorsite, a self-cleaving peptide sequence, an effector gene, and anoptional transcript stabilization element, wherein the cell comprises atarget gene differentially expressed in a unique cell type and/or in acell during a unique cell state, wherein the target gene comprises anintron comprising the recognition site, and wherein the programmablenuclease is capable of cleaving the recognition site, whereby the donornucleic acid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair, and thereby theintroducing treats the disease or disorder in the subject.

In some embodiments, the donor nucleic acid comprises a translationframe linker. In some embodiments, the donor nucleic acid comprises thestructure 5′-[recognition site]-[splice acceptor site]-[translationframe linker]-[self-cleaving peptide sequence]-[effector gene]-3′. Insome embodiments, the donor nucleic acid comprises the target genecoding sequence downstream of the intron. In some embodiments, the donornucleic acid comprises the structure 5′-[recognition site]-[spliceacceptor site]-[target gene coding sequence downstream of theintron]-[self-cleaving peptide sequence]-[effector gene]-3′. In someembodiments, the recognition site of the donor nucleic acid and therecognition site of the intron are the same. In some embodiments, therecognition site does not exist after the donor nucleic acid has beenproperly incorporated into the intron. In some embodiments, the donornucleic acid does not comprise a promoter.

In some embodiments, the effector gene is not expressed in a cell otherthan the unique cell type. In some embodiments, the expression of theeffector gene in a cell other than the unique cell type is less thanabout 5 percent of the expression of the effector gene in the uniquecell type. In some embodiments, the unique cell type comprises a uniquegene expression pattern. In some embodiments, the unique cell typecomprises a unique anatomic location. In some embodiments, the uniquecell type comprises anatomically locally unique gene expression. In someembodiments, the effector gene is not expressed in a cell other than ina cell during the unique cell state. In some embodiments, the expressionof the effector gene in a cell not in the unique cell state is less thanabout 10 percent of the expression of the effector gene in a cell duringthe unique cell state. In some embodiments, the effector gene is onlyexpressed in a cell expressing the target gene. In some embodiments, theexpression of the effector gene in a cell that does not express thetarget gene is less than about 5 percent of the expression of theeffector gene in a cell that does express the target gene. In someembodiments, the unique cell state is caused by hereditable,environmental, and/or idiopathic factors.

In some embodiments, the cell comprises a plurality of cells. In someembodiments, the donor nucleic acid is capable of being incorporatedinto the intron of a post-mitotic cell. In some embodiments, the cell isa dividing cell, a non-dividing cell, a post-mitotic cell, or anycombination thereof. In some embodiments, the cell is not in G2/M phase.In some embodiments, the cell is a eukaryotic cell (e.g., an immunecell, an epithelial cell, a muscle cell, an endothelial cell, a neuron,a stem cell, or any combination thereof) In some embodiments, the immunecell comprises a T cell, a B cell, a natural killer cell, a monocyte, amacrophage cell, a dendritic cell, or any combination thereof. In someembodiments, the stem cell comprises an embryonic stem cell, an inducedpluripotent stem cell (iPSC), a hematopoietic stem/progenitor cell(HSPC), or any combination thereof. In some embodiments, the cell, theunique cell type and/or the cell in the unique cell state comprises aneuron. In some embodiments, the cell, the unique cell type and/or thecell in the unique cell state comprises a cell in a brain region. Insome embodiments, the neuron is: involved in memory expression, involvedin eating control, involved in addiction, a component of a motor controlcircuit, an anxiety processing neuron, an analgesia inducing neuron, ananalgesia processing neuron, a pain-processing neuron, or anycombination thereof. In some embodiments, the neuron is associated witha neurological disease or disorder. In some embodiments, the neuron cancompensate, reverse or alleviate a disorder/disease state. In someembodiments, the unique cell type and/or the cell in the unique cellstate causes and/or aggravates a disease or disorder. In someembodiments, the unique cell type and/or the cell in the unique cellstate is associated with the pathology of a disease or disorder. In someembodiments, the cell is the cell of a subject. In some embodiments, thecell is the cell of a subject suffering from a disease or disorder.

In some embodiments, the disease or disorder is a blood disease, animmune disease, a cancer, an infectious disease, a genetic disease, adisorder caused by aberrant mtDNA, a metabolic disease, a disordercaused by aberrant cell cycle, a disorder caused by aberrantangiogenesis, a disorder cause by aberrant DNA damage repair, or anycombination thereof. In some embodiments, the disease or disordercomprises a neurological disease or disorder. In some embodiments, theneurological disease or disorder comprises Alzheimer's disease,Creutzfeld-Jakob's syndrome/disease, bovine spongiform encephalopathy(BSE), prion related infections, diseases involving mitochondrialdysfunction, diseases involving β-amyloid and/or tauopathy, Down'ssyndrome, hepatic encephalopathy, Huntington's disease, motor neurondiseases, amyotrophic lateral sclerosis (ALS), olivoponto-cerebellaratrophy, post-operative cognitive deficit (POCD), systemic lupuserythematosus, systemic clerosis, Sjogren's syndrome, Neuronal CeroidLipofuscinosis, neurodegenerative cerebellar ataxias, Parkinson'sdisease, Parkinson's dementia, mild cognitive impairment, cognitivedeficits in various forms of mild cognitive impairment, cognitivedeficits in various forms of dementia, dementia pugilistica, vascularand frontal lobe dementia, cognitive impairment, learning impairment,eye injuries, eye diseases, eye disorders, glaucoma, retinopathy,macular degeneration, head or brain or spinal cord injuries, head orbrain or spinal cord trauma, convulsions, epileptic convulsions,epilepsy, temporal lobe epilepsy, myoclonic epilepsy, tinnitus,dyskinesias, chorea, Huntington's chorea, athetosis, dystonia,stereotypy, ballism, tardive dyskinesias, tic disorder, torticollisspasmodicus, blepharospasm, focal and generalized dystonia, nystagmus,hereditary cerebellar ataxias, corticobasal degeneration, tremor,essential tremor, addiction, anxiety disorders, panic disorders, socialanxiety disorder (SAD), attention deficit hyperactivity disorder (ADHD),attention deficit syndrome (ADS), restless leg syndrome (RLS),hyperactivity in children, autism, dementia, dementia in Alzheimer'sdisease, dementia in Korsakoff syndrome, Korsakoff syndrome, vasculardementia, dementia related to HIV infections, HIV-1 encephalopathy, AIDSencephalopathy, AIDS dementia complex, AIDS-related dementia, majordepressive disorder, major depression, depression, memory loss, stress,bipolar manic-depressive disorder, drug tolerance, drug tolerance toopioids, movement disorders, fragile-X syndrome, irritable bowelsyndrome (IBS), migraine, multiple sclerosis (MS), muscle spasms, pain,chronic pain, acute pain, inflammatory pain, neuropathic pain,posttraumatic stress disorder (PTSD), schizophrenia, spasticity,Tourette's syndrome, eating disorders, food addiction, binge eatingdisorders, agoraphobia, generalized anxiety disorder,obsessive-compulsive disorder, panic disorder, social phobia, phobicdisorders, substance-induced anxiety disorder, delusional disorder,schizoaffective disorder, schizophreniform disorder, substance-inducedpsychotic disorder, hypertension, or any combination thereof. In someembodiments, the neurological disease or disorder comprises aneuro-psychiatric disorder.

In some embodiments, the neurological disease or disorder comprisespain. In some embodiments, the neurological disease or disordercomprises acute pain and/or chronic pain. In some embodiments, the painis selected from the group comprising neuropathic pain, allodynia,hyperalgesia, dysesthesia, causalgia, neuralgia, and arthralgia. In someembodiments, the pain is associated with cancer, tumor pressure, bonemetastasis, chemotherapy peripheral neuropathy, sciatica radiculopathy,lumbar radiculopathy, cervical radiculopathy, failed back surgerysyndrome, piriformis syndrome, phantom pain, arachnoiditis,fibromyalgia, facet joint mediated pain, sympathetically-mediated painsyndrome, complex regional pain syndromes (crps), sacroiliac (si) jointmediated pain, meralgia paresthetica, localized myofacial painsyndromes, myofacial trigger points, diffuse myofacial pain syndrome,post-herpetic neuralgia, trigeminal neuralgia, glossopharyngealneuralgia, post-epesiotomy scar pain, post-hernia repair scar pain,post-surgery scar pain, post-radiotherapy scar pain, vulvodynia,vaginismus, levator ani syndrome, chronic prostatitis, interstitialcystitis, first bite syndrome, rheumatoid arthritis pain, osteoarthritispain, atypical odontalgia, phantom tooth pain, neuropathic orofacialpain, primary erythermalgia, atypical facial pain, or any combinationthereof.

In some embodiments, the expression of the effector gene is capable ofmodulating cellular membrane potential. In some embodiments, theexpression of the effector gene is capable of altering the membranepotential of the cell by depolarizing the cell and/or hyperpolarizingthe cell. In some embodiments, the expression of the effector gene iscapable of reducing synaptic transmission by at least 10 percent. Insome embodiments, the expression of the effector gene is capable ofblocking synaptic transmission. In some embodiments, the effector genecomprises Kir2.1. In some embodiments, the expression of the effectorgene is capable of blocking synaptic transmission. In some embodiments,the effector gene comprises tetanus toxin (TNT). In some embodiments,the expression of the effector gene is capable of sensitizing the cellto a pharmacological compound, temperature change, or light. In someembodiments, the effector gene comprises a DREADD receptor and/orChannelrhodopsin-2. In some embodiments, the effector gene is capable ofsensitizing the cell to a drug and/or to a prodrug. In some embodiments,the effector gene comprises cytosine deaminase and/or uracilphosphoribosyl transferase, and wherein the prodrug comprises5-fluorocytosine (5-FC). In some embodiments, the effector genecomprises thymidine kinase (TK), and wherein the prodrug comprisesganciclovir. In some embodiments, the effector gene comprises an enzyme,a signal transduction protein, an ion-channel and/or a G-protein coupledreceptor (GPCR). In some embodiments, the effector gene encodes arecombinant protein and/or a native protein. In some embodiments, theeffector gene is capable of inducing cell death. In some embodiments,the effector gene comprises cytosine deaminase, thymidine kinase, Bax,Bid, Bad, Bak, BCL2L11, p53, PUMA, Diablo/SMAC, S-TRAIL, Cas9, Cas9n,hSpCas9, hSpCas9n, HSVtk, cholera toxin, diphtheria toxin, alpha toxin,anthrax toxin, exotoxin, pertussis toxin, Shiga toxin, shiga-like toxinFas, TNF, caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase9, caspase 10, caspase 11, caspase 12, purine nucleoside phosphorylase,or any combination thereof. In some embodiments, the unique cell statecomprises a senescent cell state induced by a tumor microenvironment. Insome embodiments, the senescent cell state induced by a tumormicroenvironment comprises expression of CD57, KRLG1, TIGIT, or anycombination thereof. In some embodiments, the effector gene comprisesinterleukin-12 (IL-12). In some embodiments, the effector gene comprisesa diagnostic agent. In some embodiments, the effector gene comprises adiagnostic contrast agent. In some embodiments, the effector genecomprises green fluorescent protein (GFP), enhanced green fluorescentprotein (EGFP), yellow fluorescent protein (YFP), enhanced yellowfluorescent protein (EYFP), blue fluorescent protein (BFP), redfluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry,rsCherry, rsCherryRev, or any combination thereof.

In some embodiments, the method achieves an at least 10 percent highertargeting efficiency as compared to a homology directed repair(HDR)-based method. In some embodiments, the method achieves an at least10 percent higher targeting efficiency as compared to a HomologyIndependent Targeted Integration (HITI)-based method integrating aneffector gene into an exon. In some embodiments, the programmablenuclease is capable of inducing a double-stranded DNA break. In someembodiments, the programmable nuclease comprises Streptococcus pyogenesCas9 (SpCas9) and/or Staphylococcus aureus Cas9 (SaCas9). In someembodiments, the programmable nuclease comprises Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3,Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c,derivatives thereof, or any combination thereof. In some embodiments,the programmable nuclease comprises a zinc finger nuclease, TAL effectornuclease, meganuclease, MegaTAL, Tev-m TALEN, MegaTev, homingendonuclease, derivatives thereof, or any combination thereof.

In some embodiments, the self-cleaving peptide sequence comprisesporcine teschovirus-1 2A peptide (P2A), Thosea asigna virus 2A peptide(T2A), equine rhinitis A virus 2A peptide (E2A), foot-and-mouth diseasevirus 2A peptide (F2A), or any combination thereof. In some embodiments,the transcript stabilization element is capable of enhancing thestability of a transcript of the effector gene. In some embodiments, thetranscript stabilization element comprises woodchuck hepatitispost-translational regulatory element (WPRE), bovine growth hormonepolyadenylation (bGH-polyA) signal sequence, human growth hormonepolyadenylation (hGH-polyA) signal sequence, or any combination thereof.In some embodiments, the splice acceptor site is capable of beingrecognized and cleaved by a spliceosome. In some embodiments, the spliceacceptor site comprises a branchpoint, a polypyrimidine tract, a 3′splice site, or any combination thereof. In some embodiments, thetranslation frame linker comprises between about 1 nucleotide and about10 nucleotides and ensures that the effector gene is expressed in framewith the preceding exon of the target gene. In some embodiments, thetranslation frame linker places the effector gene in translational framewith the preceding exon of the target gene. In some embodiments, therecognition site is adjacent to a protospacer adjacent motif (PAM)capable of being recognized by the programmable nuclease. In someembodiments, the targeting molecule is capable of associating with theprogrammable nuclease. In some embodiments, the targeting moleculecomprises single strand DNA or single strand RNA. In some embodiments,the targeting molecule comprises a single guide RNA (sgRNA). In someembodiments, the targeting molecule comprises a synthetic nucleic acid.

In some embodiments, the programmable nuclease, the targeting molecule,and/or the donor nucleic acid are encoded on the same nucleic acid. Insome embodiments, the programmable nuclease, the targeting molecule,and/or the donor nucleic acid are encoded on different nucleic acids. Insome embodiments, the nucleic acid encoding the programmable nuclease,the nucleic acid encoding the targeting molecule, and/or the nucleicacid encoding the donor nucleic acid comprise DNA and/or RNA. In someembodiments, the nucleic acid encoding the programmable nuclease, thenucleic acid encoding the targeting molecule, and/or the nucleic acidencoding the donor nucleic acid are component of a vector. In someembodiments, the vector is an AAV vector, a lentivirus, anintegration-deficient lentivirus (IDLV), a plasmid vector, a naked DNAvector, a lipid nanoparticle, or any combination thereof. In someembodiments, the plasmid vector comprises a minicircle plasmid. In someembodiments, the AAV vector comprises AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, or any combination thereof. In some embodiments,the vector is a neurotropic viral vector. In some embodiments, theneurotropic viral vector comprises or is derived from Herpesviridae,varicella zoster virus, pseudorabies virus, cyromegalovirus,Epstein-barr virus, encephalitis virus, polio virus, coxsackie virus,echo virus, mumps virus, measles virus, rabies virus, or any combinationthereof. In some embodiments, the vector comprises a heterologouspromoter that drives expression of the programmable nuclease, thetargeting molecule, and/or the donor nucleic acid. In some embodiments,the heterologous promoter is an inducible promoter. In some embodiments,the programmable nuclease, the targeting molecule, and/or the donornucleic acid are encoded by a transgenic construct in the genome of thecell. In some embodiments, (i), (ii), and/or (iii) are a component of aribonucleoprotein (RNP) complex.

The method can comprise: isolating the cell from the subject prior tothe introducing step. The method can comprise: administering the cellinto a subject after the introducing step. In some embodiments, theintroducing step is performed in vivo, in vitro, and/or ex vivo. In someembodiments, the introducing step comprises calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, electrical nucleartransport, chemical transduction, electrotransduction,Lipofectamine-mediated transfection, Effectene-mediated transfection,lipid nanoparticle (LNP)-mediated transfection, or any combinationthereof. In some embodiments, the introducing step comprisesadministering (i), (ii), and/or (iii) to a subject comprising the cell.In some embodiments, the introducing step comprises administering avector to a subject comprising the cell, wherein the vector comprises(i), (ii), and/or (iii). In some embodiments, administering comprisesintracranial injection, aerosol delivery, nasal delivery, vaginaldelivery, rectal delivery, buccal delivery, ocular delivery, localdelivery, topical delivery, intracisternal delivery, intraperitonealdelivery, oral delivery, intramuscular injection, intravenous injection,subcutaneous injection, intranodal injection, intratumoral injection,intraperitoneal injection, and/or intradermal injection, or anycombination thereof. In some embodiments, administering comprises aninjection into a brain region. In some embodiments, the brain regioncomprises the Lateral parabrachial nucleus, brainstem, Medullaoblongata, Medullary pyramids, Olivary body, Inferior olivary nucleus,Rostral ventrolateral medulla, Respiratory center, Dorsal respiratorygroup, Ventral respiratory group, Pre-Bötzinger complex, Botzingercomplex, Paramedian reticular nucleus, Cuneate nucleus, Gracile nucleus,Intercalated nucleus, Area postrema, Medullary cranial nerve nuclei,Inferior salivatory nucleus, Nucleus ambiguus, Dorsal nucleus of vagusnerve, Hypoglossal nucleus, Solitary nucleus, Pons, Pontine nuclei,Pontine cranial nerve nuclei, chief or pontine nucleus of the trigeminalnerve sensory nucleus (V), Motor nucleus for the trigeminal nerve (V),Abducens nucleus (VI), Facial nerve nucleus (VII), vestibulocochlearnuclei (vestibular nuclei and cochlear nuclei) (VIII), Superiorsalivatory nucleus, Pontine tegmentum, Respiratory centers, Pneumotaxiccenter, Apneustic center, Pontine micturition center (Barrington'snucleus), Locus coeruleus, Pedunculopontine nucleus, Laterodorsaltegmental nucleus, Tegmental pontine reticular nucleus, Superior olivarycomplex, Paramedian pontine reticular formation, Cerebellar peduncles,Superior cerebellar peduncle, Middle cerebellar peduncle, Inferiorcerebellar peduncle, Cerebellum, Cerebellar vermis, Cerebellarhemispheres, Anterior lobe, Posterior lobe, Flocculonodular lobe,Cerebellar nuclei, Fastigial nucleus, Interposed nucleus, Globosenucleus, Emboliform nucleus, Dentate nucleus, Tectum, Corporaquadrigemina, inferior colliculi, superior colliculi, Pretectum,Tegmentum, Periaqueductal gray, Parabrachial area, Medial parabrachialnucleus, Subparabrachial nucleus (Kölliker-Fuse nucleus), Rostralinterstitial nucleus of medial longitudinal fasciculus, Midbrainreticular formation, Dorsal raphe nucleus, Red nucleus, Ventraltegmental area, Substantia nigra, Pars compacta, Pars reticulata,Interpeduncular nucleus, Cerebral peduncle, Crus cerebri, Mesencephaliccranial nerve nuclei, Oculomotor nucleus (III), Trochlear nucleus (IV),Mesencephalic duct (cerebral aqueduct, aqueduct of Sylvius), Pinealbody, Habenular nucleim Stria medullares, Taenia thalami, Subcommissuralorgan, Thalamus, Anterior nuclear group, Anteroventral nucleus (akaventral anterior nucleus), Anterodorsal nucleus, Anteromedial nucleus,Medial nuclear group, Medial dorsal nucleus, Midline nuclear group,Paratenial nucleus, Reuniens nucleus, Rhomboidal nucleus, Intralaminarnuclear group, Centromedial nucleus, Parafascicular nucleus, Paracentralnucleus, Central lateral nucleus, Central medial nucleus, Lateralnuclear group, Lateral dorsal nucleus, Lateral posterior nucleus,Pulvinar, Ventral nuclear group, Ventral anterior nucleus, Ventrallateral nucleus, Ventral posterior nucleus, Ventral posterior lateralnucleus, Ventral posterior medial nucleus, Metathalamus, Medialgeniculate body, Lateral geniculate body, Thalamic reticular nucleus,Hypothalamus, limbic system, HPA axis, preoptic area, Medial preopticnucleus, Suprachiasmatic nucleus, Paraventricular nucleus, Supraopticnucleusm Anterior hypothalamic nucleus, Lateral preoptic nucleus, medianpreoptic nucleus, periventricular preoptic nucleus, Tuberal, Dorsomedialhypothalamic nucleus, Ventromedial nucleus, Arcuate nucleus, Lateralarea, Tuberal part of Lateral nucleus, Lateral tuberal nuclei,Mammillary nuclei, Posterior nucleus, Lateral area, Optic chiasm,Subfornical organ, Periventricular nucleus, Pituitary stalk, Tubercinereum, Tuberal nucleus, Tuberomammillary nucleus, Tuberal region,Mammillary bodies, Mammillary nucleus, Subthalamus, Subthalamic nucleus,Zona incerta, Pituitary gland, neurohypophysis, Pars intermedia,adenohypophysis, cerebral hemispheres, Corona radiata, Internal capsule,External capsule, Extreme capsule, Arcuate fasciculus, Uncinatefasciculus, Perforant Path, Hippocampus, Dentate gyms, Cornu ammonis,Cornu ammonis area 1, Cornu ammonis area 2, Cornu ammonis area 3, Cornuammonis area 4, Amygdala, Central nucleus, Medial nucleus (accessoryolfactory system), Cortical and basomedial nuclei, Lateral andbasolateral nuclei, extended amygdala, Stria terminalis, Bed nucleus ofthe stria terminalis, Claustrum, Basal ganglia, Striatum, Dorsalstriatum (aka neostriatum), Putamen, Caudate nucleus, Ventral striatum,Striatum, Nucleus accumbens, Olfactory tubercle, Globus pallidus,Subthalamic nucleus, Basal forebrain, Anterior perforated substance,Substantia innominata, Nucleus basalis, Diagonal band of Broca, Septalnuclei, Medial septal nuclei, Lamina terminalis, Vascular organ oflamina terminalis, Olfactory bulb, Piriform cortex, Anterior olfactorynucleus, Olfactory tract, Anterior commissure, Uncus, Cerebral cortex,Frontal lobe, Frontal cortex, Primary motor cortex, Supplementary motorcortex, Premotor cortex, Prefrontal cortex, frontopolar cortex,Orbitofrontal cortex, Dorsolateral prefrontal cortex, dorsomedialprefrontal cortex, ventrolateral prefrontal cortex, Superior frontalgyms, Middle frontal gyms, Inferior frontal gyms, Brodmann areas (4, 6,8, 9, 10, 11, 12, 24, 25, 32, 33, 44, 45, 46, and/or 47), Parietal lobe,Parietal cortex, Primary somatosensory cortex (S1), Secondarysomatosensory cortex (S2), Posterior parietal cortex, postcentral gyms,precuneus, Brodmann areas (1, 2, 3 (Primary somesthetic area), 5, 7, 23,26, 29, 31, 39, and/or 40), Occipital lobe, Primary visual cortex (V1),V2, V3, V4, V5/MT, Lateral occipital gyms, Cuneus, Brodmann areas (17(V1, primary visual cortex), 18, and/or 19), temporal lobe, Primaryauditory cortex (A1), secondary auditory cortex (A2), Inferior temporalcortex, Posterior inferior temporal cortex, Superior temporal gyms,Middle temporal gyms, Inferior temporal gyms, Entorhinal Cortex,Perirhinal Cortex, Parahippocampal gyms, Fusiform gyms, Brodmann areas(9, 20, 21, 22, 27, 34, 35, 36, 37, 38, 41, and/or 42), Medial superiortemporal area (MST), insular cortex, cingulate cortex, Anteriorcingulate, Posterior cingulate, dorsal cingulate, Retrosplenial cortex,Indusium griseum, Subgenual area 25, Brodmann areas (23, 24; 26, 29, 30(retrosplenial areas), 31, and/or 32), cranial nerves (Olfactory (I),Optic (II), Oculomotor (III), Trochlear (IV), Trigeminal (V), Abducens(VI), Facial (VII), Vestibulocochlear (VIII), Glossopharyngeal (IX),Vagus (X), Accessory (XI), Hypoglossal (XII)), or any combinationthereof. In some embodiments, the brain region comprises neural pathwaysSuperior longitudinal fasciculus, Arcuate fasciculus, Thalamocorticalradiations, Cerebral peduncle, Corpus callosum, Posterior commissure,Pyramidal or corticospinal tract, Medial longitudinal fasciculus,dopamine system, Mesocortical pathway, Mesolimbic pathway, Nigrostriatalpathway, Tuberoinfundibular pathway, serotonin system, NorepinephrinePathways, Posterior column-medial lemniscus pathway, Spinothalamictract, Lateral spinothalamic tract, Anterior spinothalamic tract, or anycombination thereof.

Disclosed herein include compositions. In some embodiments, thecomposition comprises: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and/or (iii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid, whereinthe donor nucleic acid comprises a recognition site, a splice acceptorsite, a self-cleaving peptide sequence, an effector gene, and anoptional transcript stabilization element, and wherein the targetingmolecule is complementary to the recognition site.

Disclosed herein include compositions. In some embodiments, thecomposition comprises: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease, wherein the programmable nucleasecomprises a zinc finger nuclease (ZFN) and/or transcriptionactivator-like effector nuclease (TALEN); and/or (ii) a donor nucleicacid or a nucleic acid encoding the donor nucleic acid, wherein thedonor nucleic acid comprises a recognition site, a splice acceptor site,a self-cleaving peptide sequence, an effector gene, and an optionaltranscript stabilization element.

In some embodiments, a cell of a subject comprises a target genedifferentially expressed in a unique cell type and/or in a cell during aunique cell state, wherein the target gene comprises an introncomprising the recognition site. In some embodiments, the programmablenuclease is capable of cleaving the recognition site, whereby the donornucleic acid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair. In someembodiments, the donor nucleic acid comprises a translation framelinker. In some embodiments, the donor nucleic acid comprises thestructure 5′-[recognition site]-[splice acceptor site]-[translationframe linker]-[self-cleaving peptide sequence]-[effector gene]-3′. Insome embodiments, the donor nucleic acid comprises the target genecoding sequence downstream of the intron. In some embodiments, the donornucleic acid comprises the structure 5′-[recognition site]-[spliceacceptor site]-[target gene coding sequence downstream of theintron]-[self-cleaving peptide sequence]-[effector gene]-3′. In someembodiments, the donor nucleic acid does not comprise a transcriptstabilization element. In some embodiments, the donor nucleic acidcomprises a transcript stabilization element. The donor nucleic acid cancomprise the structure 5′-[recognition site]-[splice acceptorsite]-[translation frame linker]-[self-cleaving peptidesequence]-[effector gene]-[transcript stabilization element]-3′. Thedonor nucleic acid can comprise the structure 5′-[recognitionsite]-[splice acceptor site]-[target gene coding sequence downstream ofthe intron]-[self-cleaving peptide sequence]-[transcript stabilizationelement]-3′. In some embodiments, the recognition site of the donornucleic acid and the recognition site of the intron are the same. Insome embodiments, the recognition site of the donor nucleic acid and therecognition site of the intron share at least 70 percent sequenceidentity. In some embodiments, the recognition site does not exist afterthe donor nucleic acid has been properly incorporated into the intron.In some embodiments, the donor nucleic acid does not comprise apromoter.

In some embodiments, the effector gene is not expressed in a cell otherthan the unique cell type. In some embodiments, the expression of theeffector gene in a cell other than the unique cell type is less thanabout 5 percent of the expression of the effector gene in the uniquecell type. In some embodiments, the unique cell type comprises a uniquegene expression pattern. In some embodiments, the unique cell typecomprises a unique anatomic location. In some embodiments, the uniquecell type comprises anatomically locally unique gene expression. In someembodiments, the effector gene is not expressed in a cell other than ina cell during the unique cell state. In some embodiments, the expressionof the effector gene in a cell not in the unique cell state is less thanabout 10 percent of the expression of the effector gene in a cell duringthe unique cell state. In some embodiments, the effector gene is onlyexpressed in a cell expressing the target gene. In some embodiments, theexpression of the effector gene in a cell that does not express thetarget gene is less than about 5 percent of the expression of theeffector gene in a cell that does express the target gene. In someembodiments, the unique cell state is caused by hereditable,environmental, and/or idiopathic factors.

In some embodiments, the cell comprises a plurality of cells. In someembodiments, the donor nucleic acid is capable of being incorporatedinto the intron of a post-mitotic cell. In some embodiments, the cell isa dividing cell, a non-dividing cell, a post-mitotic cell, or anycombination thereof. In some embodiments, the cell is not in G2/M phase.In some embodiments, the cell is a eukaryotic cell. In some embodiments,the eukaryotic cell comprises an immune cell, a muscle cell, anepithelial cell, an endothelial cell, a neuron, a stem cell, or anycombination thereof. In some embodiments, the immune cell comprises a Tcell, a B cell, a natural killer cell, a monocyte, a macrophage cell, adendritic cell, or any combination thereof. In some embodiments, thestem cell comprises an embryonic stem cell, an induced pluripotent stemcell (iPSC), a hematopoietic stem/progenitor cell (HSPC), or anycombination thereof. In some embodiments, the cell, the unique cell typeand/or the cell in the unique cell state comprises a neuron. In someembodiments, the neuron is: involved in memory expression, involved ineating control, involved in addiction, a component of a motor controlcircuit, an anxiety processing neuron, an analgesia inducing neuron, ananalgesia processing neuron, a pain-processing neuron, or anycombination thereof. In some embodiments, the neuron is associated witha neurological disease or disorder. In some embodiments, the neuron cancompensate, reverse or alleviate a disorder/disease state. In someembodiments, the unique cell type and/or the cell in the unique cellstate causes and/or aggravates a disease or disorder. In someembodiments, the unique cell type and/or the cell in the unique cellstate is associated with the pathology of a disease or disorder. In someembodiments, the cell is the cell of a subject.

In some embodiments, the cell is the cell of a subject suffering from adisease or disorder. In some embodiments, the disease or disorder is ablood disease, an immune disease, a cancer, an infectious disease, agenetic disease, a disorder caused by aberrant mtDNA, a metabolicdisease, a disorder caused by aberrant cell cycle, a disorder caused byaberrant angiogenesis, a disorder cause by aberrant DNA damage repair,or any combination thereof. In some embodiments, the disease or disordercomprises a neurological disease or disorder. In some embodiments, theneurological disease or disorder comprises Alzheimer's disease,Creutzfeld-Jakob's syndrome/disease, bovine spongiform encephalopathy(BSE), prion related infections, diseases involving mitochondrialdysfunction, diseases involving β-amyloid and/or tauopathy, Down'ssyndrome, hepatic encephalopathy, Huntington's disease, motor neurondiseases, amyotrophic lateral sclerosis (ALS), olivoponto-cerebellaratrophy, post-operative cognitive deficit (POCD), systemic lupuserythematosus, systemic clerosis, Sjogren's syndrome, Neuronal CeroidLipofuscinosis, neurodegenerative cerebellar ataxias, Parkinson'sdisease, Parkinson's dementia, mild cognitive impairment, cognitivedeficits in various forms of mild cognitive impairment, cognitivedeficits in various forms of dementia, dementia pugilistica, vascularand frontal lobe dementia, cognitive impairment, learning impairment,eye injuries, eye diseases, eye disorders, glaucoma, retinopathy,macular degeneration, head or brain or spinal cord injuries, head orbrain or spinal cord trauma, convulsions, epileptic convulsions,epilepsy, temporal lobe epilepsy, myoclonic epilepsy, tinnitus,dyskinesias, chorea, Huntington's chorea, athetosis, dystonia,stereotypy, ballism, tardive dyskinesias, tic disorder, torticollisspasmodicus, blepharospasm, focal and generalized dystonia, nystagmus,hereditary cerebellar ataxias, corticobasal degeneration, tremor,essential tremor, addiction, anxiety disorders, panic disorders, socialanxiety disorder (SAD), attention deficit hyperactivity disorder (ADHD),attention deficit syndrome (ADS), restless leg syndrome (RLS),hyperactivity in children, autism, dementia, dementia in Alzheimer'sdisease, dementia in Korsakoff syndrome, Korsakoff syndrome, vasculardementia, dementia related to HIV infections, HIV-1 encephalopathy, AIDSencephalopathy, AIDS dementia complex, AIDS-related dementia, majordepressive disorder, major depression, depression, memory loss, stress,bipolar manic-depressive disorder, drug tolerance, drug tolerance toopioids, movement disorders, fragile-X syndrome, irritable bowelsyndrome (IBS), migraine, multiple sclerosis (MS), muscle spasms, pain,chronic pain, acute pain, inflammatory pain, neuropathic pain,posttraumatic stress disorder (PTSD), schizophrenia, spasticity,Tourette's syndrome, eating disorders, food addiction, binge eatingdisorders, agoraphobia, generalized anxiety disorder,obsessive-compulsive disorder, panic disorder, social phobia, phobicdisorders, substance-induced anxiety disorder, delusional disorder,schizoaffective disorder, schizophreniform disorder, substance-inducedpsychotic disorder, hypertension, or any combination thereof. In someembodiments, the neurological disease or disorder comprises aneuro-psychiatric disorder.

In some embodiments, the neurological disease or disorder comprisespain. In some embodiments, the neurological disease or disordercomprises acute pain and/or chronic pain. In some embodiments, the painis selected from the group comprising neuropathic pain, allodynia,hyperalgesia, dysesthesia, causalgia, neuralgia, and arthralgia. In someembodiments, the pain is associated with cancer, tumor pressure, bonemetastasis, chemotherapy peripheral neuropathy, sciatica radiculopathy,lumbar radiculopathy, cervical radiculopathy, failed back surgerysyndrome, piriformis syndrome, phantom pain, arachnoiditis,fibromyalgia, facet joint mediated pain, sympathetically-mediated painsyndrome, complex regional pain syndromes (crps), sacroiliac (si) jointmediated pain, meralgia paresthetica, localized myofacial painsyndromes, myofacial trigger points, diffuse myofacial pain syndrome,post-herpetic neuralgia, trigeminal neuralgia, glossopharyngealneuralgia, post-epesiotomy scar pain, post-hernia repair scar pain,post-surgery scar pain, post-radiotherapy scar pain, vulvodynia,vaginismus, levator ani syndrome, chronic prostatitis, interstitialcystitis, first bite syndrome, rheumatoid arthritis pain, osteoarthritispain, atypical odontalgia, phantom tooth pain, neuropathic orofacialpain, primary erythermalgia, atypical facial pain, or any combinationthereof.

In some embodiments, the expression of the effector gene is capable ofmodulating cellular membrane potential. In some embodiments, theexpression of the effector gene is capable of altering the membranepotential of the cell by depolarizing the cell and/or hyperpolarizingthe cell. In some embodiments, the expression of the effector gene iscapable of reducing synaptic transmission by at least 10 percent. Insome embodiments, the expression of the effector gene is capable ofblocking synaptic transmission. In some embodiments, the effector genecomprises Kir2.1. In some embodiments, the expression of the effectorgene is capable of blocking synaptic transmission. In some embodiments,the effector gene comprises tetanus toxin (TNT). In some embodiments,the expression of the effector gene is capable of sensitizing the cellto a pharmacological compound, temperature change, or light. In someembodiments, the effector gene comprises a DREADD receptor and/orChannelrhodopsin-2. In some embodiments, the effector gene is capable ofsensitizing the cell to a drug and/or to a prodrug. In some embodiments,the effector gene comprises cytosine deaminase and/or uracilphosphoribosyl transferase, and wherein the prodrug comprises5-fluorocytosine (5-FC). In some embodiments, the effector genecomprises thymidine kinase (TK), and wherein the prodrug comprisesganciclovir. In some embodiments, the effector gene comprises an enzyme,a signal transduction protein, an ion-channel and/or a G-protein coupledreceptor (GPCR). In some embodiments, the effector gene encodes arecombinant protein and/or a native protein. In some embodiments, theeffector gene is capable of inducing cell death. In some embodiments,the effector gene comprises cytosine deaminase, thymidine kinase, Bax,Bid, Bad, Bak, BCL2L11, p53, PUMA, Diablo/SMAC, S-TRAIL, Cas9, Cas9n,hSpCas9, hSpCas9n, HSVtk, cholera toxin, diphtheria toxin, alpha toxin,anthrax toxin, exotoxin, pertussis toxin, Shiga toxin, shiga-like toxinFas, TNF, caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase9, caspase 10, caspase 11, caspase 12, purine nucleoside phosphorylase,or any combination thereof. In some embodiments, the unique cell statecomprises a senescent cell state induced by a tumor microenvironment. Insome embodiments, the senescent cell state induced by a tumormicroenvironment comprises expression of CD57, KRLG1, TIGIT, or anycombination thereof. In some embodiments, the effector gene comprisesinterleukin-12 (IL-12). In some embodiments, the effector gene comprisesa diagnostic agent. In some embodiments, the effector gene comprises adiagnostic contrast agent. In some embodiments, the effector genecomprises green fluorescent protein (GFP), enhanced green fluorescentprotein (EGFP), yellow fluorescent protein (YFP), enhanced yellowfluorescent protein (EYFP), blue fluorescent protein (BFP), redfluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry,rsCherry, rsCherryRev, or any combination thereof.

In some embodiments, upon introduction into a cell, the compositionachieves an at least 10 percent higher targeting efficiency as comparedto a homology directed repair (HDR)-based method. In some embodiments,upon introduction into a cell, the composition achieves an at least 10percent higher targeting efficiency as compared to a HomologyIndependent Targeted Integration (HITI)-based method integrating aneffector gene into an exon. In some embodiments, the programmablenuclease is capable of inducing a double-stranded DNA break. In someembodiments, the programmable nuclease comprises Streptococcus pyogenesCas9 (SpCas9) and/or Staphylococcus aureus Cas9 (SaCas9). In someembodiments, the programmable nuclease comprises Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3,Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c,derivatives thereof, or any combination thereof. In some embodiments,the programmable nuclease comprises a zinc finger nuclease, TAL effectornuclease, meganuclease, MegaTAL, Tev-m TALEN, MegaTev, homingendonuclease, derivatives thereof, or any combination thereof.

In some embodiments, the self-cleaving peptide sequence comprisesporcine teschovirus-1 2A peptide (P2A), Thosea asigna virus 2A peptide(T2A), equine rhinitis A virus 2A peptide (E2A), foot-and-mouth diseasevirus 2A peptide (F2A), or any combination thereof. In some embodiments,the transcript stabilization element is capable of enhancing thestability of a transcript of the effector gene. In some embodiments, thetranscript stabilization element comprises woodchuck hepatitispost-translational regulatory element (WPRE), bovine growth hormonepolyadenylation (bGH-polyA) signal sequence, human growth hormonepolyadenylation (hGH-polyA) signal sequence, or any combination thereof.In some embodiments, the splice acceptor site is capable of beingrecognized and cleaved by a spliceosome. In some embodiments, the spliceacceptor site comprises a branchpoint, a polypyrimidine tract, a 3′splice site, or any combination thereof. In some embodiments, thetranslation frame linker comprises between about 1 nucleotide and about10 nucleotides and ensures that the effector gene is expressed in framewith the preceding exon of the target gene. In some embodiments, thetranslation frame linker places the effector gene in translational framewith the preceding exon of the target gene. In some embodiments, therecognition site is adjacent to a protospacer adjacent motif (PAM)capable of being recognized by the programmable nuclease. In someembodiments, the targeting molecule is capable of associating with theprogrammable nuclease. In some embodiments, the targeting moleculecomprises single strand DNA or single strand RNA. In some embodiments,the targeting molecule comprises a single guide RNA (sgRNA). In someembodiments, the targeting molecule comprises a synthetic nucleic acid.

In some embodiments, the programmable nuclease, the targeting molecule,and/or the donor nucleic acid are encoded on the same nucleic acid. Insome embodiments, the programmable nuclease, the targeting molecule,and/or the donor nucleic acid are encoded on different nucleic acids. Insome embodiments, the nucleic acid encoding the programmable nuclease,the nucleic acid encoding the targeting molecule, and/or the nucleicacid encoding the donor nucleic acid comprise DNA and/or RNA. In someembodiments, the nucleic acid encoding the programmable nuclease, thenucleic acid encoding the targeting molecule, and/or the nucleic acidencoding the donor nucleic acid are component of a vector. In someembodiments, the vector is an AAV vector, a lentivirus, anintegration-deficient lentivirus (IDLV), a plasmid vector, a naked DNAvector, a lipid nanoparticle, or any combination thereof. In someembodiments, the plasmid vector comprises a minicircle plasmid. In someembodiments, the AAV vector comprises AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, or any combination thereof. In some embodiments,the vector is a neurotropic viral vector. In some embodiments, theneurotropic viral vector comprises or is derived from Herpesviridae,varicella zoster virus, pseudorabies virus, cyromegalovirus,Epstein-barr virus, encephalitis virus, polio virus, coxsackie virus,echo virus, mumps virus, measles virus, rabies virus, or any combinationthereof. In some embodiments, the vector comprises a heterologouspromoter that drives expression of the programmable nuclease, thetargeting molecule, and/or the donor nucleic acid. In some embodiments,the heterologous promoter is an inducible promoter. In some embodiments,(i), (ii), and/or (iii) are a component of a ribonucleoprotein (RNP)complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting exemplary schematic illustration of theCell-SELECT method for cell-type-specific expression of effector genes.

FIG. 2 shows a non-limiting exemplary schematic illustration of theCell-SELECT methods and compositions provided herein.

FIGS. 3A-3B show non-limiting exemplary schematic illustrations ofCell-SELECT donor constructs (e.g., donor nucleic acids) for cell typespecific expression of effector genes with their respective structuralelements.

FIGS. 4A-4E depict data related to the ability of the Cell-SELECT methodto target the expression of the effector gene to molecularly definedcell types in cell culture.

FIGS. 5A-5C depict data related to the knock-in efficiency of theCell-SELECT method as compared to other gene editing methods in a cellculture model expressing the targeted Tubb3 gene (N2A cell line).

FIGS. 6A-6C depict data related to in vivo gene editing in live animals.

FIGS. 7A-7B depict data related to cell targeting efficiency ofTubb3-expressing neurons with in vivo gene editing based effector geneknock-in.

FIGS. 8A-8C depict single cell RNA-seq data in the major mammalian brainpain relay—lateral parabrachial nucleus (LPBN).

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein and made part of the disclosure herein.

All patents, published patent applications, other publications, andsequences from GenBank, and other databases referred to herein areincorporated by reference in their entirety with respect to the relatedtechnology.

Disclosed herein include methods of incorporating an effector gene intothe genome of a cell. In some embodiments, the method comprises:introducing into a cell: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and (iii) a donor nucleicacid or a nucleic acid encoding the donor nucleic acid, wherein thedonor nucleic acid comprises a recognition site, a splice acceptor site,a self-cleaving peptide sequence, an effector gene, and an optionaltranscript stabilization element, wherein the cell comprises a targetgene differentially expressed in a unique cell type and/or in a cellduring a unique cell state, wherein the target gene comprises an introncomprising the recognition site, and wherein the targeting molecule iscomplementary to the recognition site and the programmable nuclease iscapable of cleaving the recognition site, whereby the donor nucleic acidis capable of being incorporated into the intron through non-homologousend joining (NHEJ)-dependent DNA repair.

Disclosed herein include methods of incorporating an effector gene intothe genome of a cell. In some embodiments, the method comprises:introducing into a cell: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease, wherein the programmable nucleasecomprises a zinc finger nuclease (ZFN) and/or transcriptionactivator-like effector nuclease (TALEN); and (ii) a donor nucleic acidor a nucleic acid encoding the donor nucleic acid, wherein the donornucleic acid comprises a recognition site, a splice acceptor site, aself-cleaving peptide sequence, an effector gene, and an optionaltranscript stabilization element, wherein the cell comprises a targetgene differentially expressed in a unique cell type and/or in a cellduring a unique cell state, wherein the target gene comprises an introncomprising the recognition site, and wherein the programmable nucleaseis capable of cleaving the recognition site, whereby the donor nucleicacid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair.

Disclosed herein include methods of treating a disease or disorder in asubject. In some embodiments, the method comprises: introducing into acell of a subject in need thereof: (i) a programmable nuclease or anucleic acid encoding the programmable nuclease; (ii) a targetingmolecule or a nucleic acid encoding the targeting molecule, and (iii) adonor nucleic acid or a nucleic acid encoding the donor nucleic acid,wherein the donor nucleic acid comprises a recognition site, a spliceacceptor site, a self-cleaving peptide sequence, an effector gene, andan optional transcript stabilization element, wherein the cell comprisesa target gene differentially expressed in a unique cell type and/or in acell during a unique cell state, wherein the target gene comprises anintron comprising the recognition site, and wherein the targetingmolecule is complementary to the recognition site and programmablenuclease is capable of cleaving the recognition site, whereby the donornucleic acid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair, and thereby theintroducing treats the disease or disorder in the subject.

Disclosed herein include methods of treating a disease or disorder in asubject. In some embodiments, the method comprises: introducing into acell of a subject in need thereof: (i) a programmable nuclease or anucleic acid encoding the programmable nuclease, wherein theprogrammable nuclease comprises a zinc finger nuclease (ZFN) and/ortranscription activator-like effector nuclease (TALEN); and (ii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid, whereinthe donor nucleic acid comprises a recognition site, a splice acceptorsite, a self-cleaving peptide sequence, an effector gene, and anoptional transcript stabilization element, wherein the cell comprises atarget gene differentially expressed in a unique cell type and/or in acell during a unique cell state, wherein the target gene comprises anintron comprising the recognition site, and wherein the programmablenuclease is capable of cleaving the recognition site, whereby the donornucleic acid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair, and thereby theintroducing treats the disease or disorder in the subject.

Disclosed herein include compositions. In some embodiments, thecomposition comprises: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and/or (iii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid, whereinthe donor nucleic acid comprises a recognition site, a splice acceptorsite, a self-cleaving peptide sequence, an effector gene, and anoptional transcript stabilization element, and wherein the targetingmolecule is complementary to the recognition site.

Disclosed herein include compositions. In some embodiments, thecomposition comprises: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease, wherein the programmable nucleasecomprises a zinc finger nuclease (ZFN) and/or transcriptionactivator-like effector nuclease (TALEN); and/or (ii) a donor nucleicacid or a nucleic acid encoding the donor nucleic acid, wherein thedonor nucleic acid comprises a recognition site, a splice acceptor site,a self-cleaving peptide sequence, an effector gene, and an optionaltranscript stabilization element.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. See, e.g. Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.1989). For purposes of the present disclosure, the following terms aredefined below.

As used herein, the term “vector” shall be given its ordinary meaning,and shall also refer to a polynucleotide construct, typically a plasmidor a virus, used to transmit genetic material to a host cell (e.g., atarget cell). Vectors can be, for example, viruses, plasmids, cosmids,or phage. A vector can be a viral vector. A vector can be aribonucleoprotein (RNP) complex. A vector as used herein can be composedof either DNA or RNA. In some embodiments, a vector is composed of DNA.An “expression vector” is a vector that is capable of directing theexpression of a protein encoded by one or more genes carried by thevector when it is present in the appropriate environment. Vectors arepreferably capable of autonomous replication. Typically, an expressionvector comprises a transcription promoter, a gene, and a transcriptionterminator. Gene expression is usually placed under the control of apromoter, and a gene is said to be “operably linked to” the promoter.

As used herein, the term “operably linked” is used to describe theconnection between regulatory elements and a gene or its coding region.Typically, gene expression is placed under the control of one or moreregulatory elements, for example, without limitation, constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.A gene or coding region is said to be “operably linked to” or“operatively linked to” or “operably associated with” the regulatoryelements, meaning that the gene or coding region is controlled orinfluenced by the regulatory element. For instance, a promoter isoperably linked to a coding sequence if the promoter effectstranscription or expression of the coding sequence.

The term “construct,” as used herein, can refer to a recombinant nucleicacid that has been generated for the purpose of the expression of aspecific nucleotide sequence(s), or that is to be used in theconstruction of other recombinant nucleotide sequences.

As used herein, the terms “nucleic acid” and “polynucleotide” areinterchangeable and refer to any nucleic acid, whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sultone linkages, andcombinations of such linkages. The terms “nucleic acid” and“polynucleotide” also specifically include nucleic acids composed ofbases other than the five biologically occurring bases (adenine,guanine, thymine, cytosine and uracil).

The terms “regulatory element” and “expression control element” are usedinterchangeably and refer to nucleic acid molecules that can influencethe expression of an operably linked coding sequence in a particularhost organism. These terms are used broadly to and cover all elementsthat promote or regulate transcription, including promoters, coreelements required for basic interaction of RNA polymerase andtranscription factors, upstream elements, enhancers, and responseelements (see e.g., Lewin, “Genes V” (Oxford University Press, Oxford)pages 847-873). Exemplary regulatory elements in prokaryotes includepromoters, operator sequences and ribosome binding sites. Regulatoryelements that are used in eukaryotic cells can include, withoutlimitation, transcriptional and translational control sequences, such aspromoters, enhancers, splicing signals, polyadenylation signals,terminators, protein degradation signals, internal ribosome-entryelement (IRES), 2A sequences, and the like, that provide for and/orregulate expression of a coding sequence and/or production of an encodedpolypeptide in a host cell.

As used herein, self-cleaving peptide sequences or elements (e.g., 2Asequences) shall be given their ordinary meaning and shall also refer tosmall peptides introduced as a linker between two polypeptides, allowingautonomous intraribosomal self-processing of polyproteins (See e.g., deFelipe. Genetic Vaccines and Ther. 2: 13 (2004); de Felipe et al.Traffic 5:616-626 (2004)). Many 2A elements are known in the art.Examples of 2A sequences that can be used in the methods and systemdisclosed herein, without limitation, include 2A sequences from thefoot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A),Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A).

As used herein, the term “promoter” is a nucleotide sequence thatpermits binding of RNA polymerase and directs the transcription of agene. Typically, a promoter is located in the 5′ non-coding region of agene, proximal to the transcriptional start site of the gene. Sequenceelements within promoters that function in the initiation oftranscription are often characterized by consensus nucleotide sequences.Examples of promoters include, but are not limited to, promoters frombacteria, yeast, plants, viruses, and mammals (including humans). Apromoter can be inducible, repressible, and/or constitutive. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asa change in temperature.

As used herein, the term “enhancer” refers to a type of regulatoryelement that can increase the efficiency of transcription, regardless ofthe distance or orientation of the enhancer relative to the start siteof transcription.

As used herein, the term “variant” refers to a polynucleotide (orpolypeptide) having a sequence substantially similar to a referencepolynucleotide (or polypeptide). In the case of a polynucleotide, avariant can have deletions, substitutions, additions of one or morenucleotides at the 5′ end, 3′ end, and/or one or more internal sites incomparison to the reference polynucleotide. Similarities and/ordifferences in sequences between a variant and the referencepolynucleotide can be detected using conventional techniques known inthe art, for example polymerase chain reaction (PCR) and hybridizationtechniques. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis. Generally, a variant of a polynucleotide,including, but not limited to, a DNA, can have at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or more sequence identity to thereference polynucleotide as determined by sequence alignment programsknown by skilled artisans. In the case of a polypeptide, a variant canhave deletions, substitutions, additions of one or more amino acids incomparison to the reference polypeptide. Similarities and/or differencesin sequences between a variant and the reference polypeptide can bedetected using conventional techniques known in the art, for exampleWestern blot. Generally, a variant of a polypeptide, can have at leastabout 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99% or more sequence identity to thereference polypeptide as determined by sequence alignment programs knownby skilled artisans.

As used herein, the term “effective amount” refers to an amountsufficient to effect beneficial or desirable biological and/or clinicalresults.

As used herein, a “subject” refers to an animal that is the object oftreatment, observation or experiment. “Animal” includes cold- andwarm-blooded vertebrates and invertebrates such as fish, shellfish,reptiles, and in particular, mammals. “Mammal,” as used herein, refersto an individual belonging to the class Mammalia and includes, but notlimited to, humans, domestic and farm animals, zoo animals, sports andpet animals. Non-limiting examples of mammals include mice; rats;rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates,such as monkeys, chimpanzees and apes, and, in particular, humans. Insome embodiments, the mammal is a human. However, in some embodiments,the mammal is not a human.

As used herein, the term “treatment” refers to an intervention made inresponse to a disease, disorder or physiological condition manifested bya patient. The aim of treatment may include, but is not limited to, oneor more of the alleviation or prevention of symptoms, slowing orstopping the progression or worsening of a disease, disorder, orcondition and the remission of the disease, disorder or condition. Theterms “treat” and “treatment” include, for example, therapeutictreatments, prophylactic treatments, and applications in which onereduces the risk that a subject will develop a disorder or other riskfactor. Treatment does not require the complete curing of a disorder andencompasses embodiments in which one reduces symptoms or underlying riskfactors. In some embodiments, “treatment” refers to both therapeutictreatment and prophylactic or preventative measures. Those in need oftreatment include those already affected by a disease or disorder orundesired physiological condition as well as those in which the diseaseor disorder or undesired physiological condition is to be prevented. Forexample, in some embodiments treatment may reduce the level of painsignaling in the subject, thereby to reduce, alleviate, or eradicate thesymptom(s) of the disease(s). As used herein, the term “prevention” canrefer to any activity that reduces the burden of the individual laterexhibiting those pain-related disease symptoms. This can take place atprimary, secondary and/or tertiary prevention levels, wherein: a)primary prevention avoids the development ofsymptoms/disorder/condition; b) secondary prevention activities areaimed at early stages of the condition/disorder/symptom treatment,thereby increasing opportunities for interventions to preventprogression of the condition/disorder/symptom and emergence of symptoms;and c) tertiary prevention reduces the negative impact of an alreadyestablished condition/disorder/symptom by, for example, restoringfunction and/or reducing any condition/disorder/symptom or relatedcomplications. The term “prevent” does not require the 100% eliminationof the possibility of an event. Rather, it denotes that the likelihoodof the occurrence of the event has been reduced in the presence of thecompound or method.

“Pharmaceutically acceptable” carriers are ones which are nontoxic tothe cell or mammal being exposed thereto at the dosages andconcentrations employed. “Pharmaceutically acceptable” carriers can be,but not limited to, organic or inorganic, solid or liquid excipientswhich is suitable for the selected mode of application such as oralapplication or injection, and administered in the form of a conventionalpharmaceutical preparation, such as solid such as tablets, granules,powders, capsules, and liquid such as solution, emulsion, suspension andthe like. Often the physiologically acceptable carrier is an aqueous pHbuffered solution such as phosphate buffer or citrate buffer. Thephysiologically acceptable carrier may also comprise one or more of thefollowing: antioxidants including ascorbic acid, low molecular weight(less than about 10 residues) polypeptides, proteins, such as serumalbumin, gelatin, immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, ammo acids, carbohydrates including glucose,mannose, or dextrins, chelating agents such as EDTA, sugar alcohols suchas mannitol or sorbitol, salt-forming counterions such as sodium, andnonionic surfactants such as Tween, polyethylene glycol (PEG), andPluronics. Auxiliary, stabilizer, emulsifier, lubricant, binder, pHadjuster controller, isotonic agent and other conventional additives mayalso be added to the carriers.

Methods and Compositions for Cellular Engineering

Disclosed herein are robust methods and compositions for precise andspecific functional manipulation of molecularly distinct cell types andstates both in in vitro and in in vivo settings. The disclosed Cell-typecontrol via Splice-capture of Expressed Locus Effector by CRISPRTargeting (Cell-SELECT) methods and compositions can comprise aneffective targeted gene knock-in approach that introduces a correctiveeffector gene into a cell-type-specifically expressed genetic locus andefficiently transcribes it by splicing it to the native transcript (FIG.1). The provided methods and compositions can either selectively ablate,inactivate, sensitize to novel compounds or otherwise functionallymanipulate distinct cell types expressing a given gene and consequentlyto specifically regulate the physiological function they execute.

A defining feature of a particular cell type or cell state can be theunique set of genes that are expressed by the cell. The disclosedCell-SELECT technology can take advantage of the genetic programs thatare active in the cell and use the cell intrinsic gene regulatorymechanisms to control selective expression of an introduced effectorgene in desired cell-types. The Cell-SELECT methods and compositionsprovided herein can ensure cell-type specific expression of thoseeffector genes by targeted intronic knock-in of the Cell-SELECT donorconstruct (e.g., donor nucleic acid). The genome integrated donornucleic acid can capture the splicing machinery duringcell-type-specifically expressed gene's transcription and can create anew hybrid transcript that expresses the effector gene in all cellswhere the target gene is expressed. Thereby, even if the genomicknock-in of the construct occurs in many cells, the effector geneexpression is only observable in target cells, where the targeted geneis actively transcribed (FIGS. 1 and 4).

The disclosed Cell-SELECT technology can rely on the cell nucleartransfer one or more of the following components: a) a Cell-SELECT donorconstruct containing the desired effector gene and functional componentsinterfacing with the cellular splicing machinery; b) Cas9 or otherprogrammable nuclease that induces double stranded breaks in the donorconstruct as well as the targeted genomic location; c) guide RNA (gRNA)or other nuclease targeting mechanism that determines the target ofnuclease activity. The Cell-SELECT donor construct can be knocked intothe intronic region of the targeted cell-type-specifically expressedtarget gene by inducing a double stranded break into the intronic areaby Cas9 and respective gRNA complex. The donor construct, that can alsoget cut and linearized by the Cas9/gRNA complex, can get integrated intothe target gene's intron by virtue of non-homologous-end-joining (NHEJ)dependent DNA repair mechanism, that happens efficiently in bothdividing as well as non-dividing cells. The right orientation of theintegrated donor construct can be ensured by reconstitution of theoriginal gRNA sites if the construct lands in the wrong orientation andhence gets recleaved (FIG. 1).

The splice capture approach implemented by some embodiments of theCell-SELECT method provided herein can achieve superior efficiency ascompared to state-of-the-art gene editing based gene tagging solutions.This makes the final cell type specific expression robust to anyimprecisions that commonly accompany NHEJ based DNA repair, variation inCas9 cleavage and degradation of the donor and genomic regions. Thisdesign feature achieves several fold higher cell targeting efficienciesas compared to currently available solutions (FIG. 6). Importantly, thisefficiency gain enables the use of Cell-SELECT based cell-type-specificfunctional targeting solutions for practically useful in vivotherapeutic and tissue engineering applications in non-transgenic adultorganisms (FIGS. 6-7). A challenge for cell type specific therapies isthe achievement of high specificity and low off-target expression ofeffector proteins. In some embodiments, the Cell-SELECT methods can beemployed for controlling the mammalian pain system.

In some embodiments, the disclosed Cell-SELECT compositions and methodsachieves cell-type specific expression of effector genes by targetedintronic knock-in of a Cell-SELECT donor construct (e.g., donor nucleicacid). The genome integrated donor can capture the splicing machineryduring cell-type-specifically expressed gene's transcription and cancreate a new hybrid transcript that expresses the effector gene in allcells where the target gene is expressed. Thereby, even though thegenomic knock-in of the construct can occur in many cells (e.g., bothtarget and non-target cells), the effector gene expression is onlyobservable in target cells, where the targeted gene is activelytranscribed (FIGS. 1 and 4). The methods and compositions providedherein yield cell-type-specific functional targeting solutions forpractically useful in vivo therapeutic and tissue engineeringapplications in non-transgenic adult organisms.

Provided herein are compositions and methods for the delivery of boththe donor and gene editing nuclease and nuclease guiding sequences(e.g., gRNA). The Cell-SELECT components disclosed herein can beintroduced to the target tissue or biological systems through non-viralmethods in the form of proteins, RNA or DNA for the gene editingmachinery and DNA template for the donor. Cell-SELECT components canalso be delivered through viral vectors, encoded by the viral genomes.In some embodiments, the disclosed Cell-SELECT approach of using cellintrinsic transcription regulatory regions to ensure cell-type-specificexpression of the effector gene obviates the need to co-deliver generegulatory regions, that are often size limiting for viral vectors andfrequently for non-viral delivery routes as well.

With the recent advent of high throughput technologies for profilinggene expression in single cells our ability to identify the geneticbasis of cell types and states has dramatically improved. Cell-SELECTtechnologies herein disclosed provides an easily deployable strategy touse these cell type and state defining genetic programs to gainfunctional control over the vast majority of cells in any organism. Thedisclosed Cell-SELECT approach overcomes the limited efficiency issuesof somatic knock-in technologies making it an attractive avenue for livetissue engineering at cell-type resolution. The Cell-SELECT methods andcompositions provided herein can comprise one or more of the followingelements to enable cell-type-specific expression of desired effectorgenes through gene editing: (1) a Cell-SELECT knock-in donor construct;(2) programmable gene editing reagents (e.g., gRNA or other nucleasetargeting mechanism and/or Cas9 or other programmable nuclease); and (3)delivery reagents (e.g., vectors).

The Cell-SELECT methods and compositions provided herein can be used tofunctionally manipulate any cell type or cell state of interest if ithas at least one transcribed gene that differentiates it from otherphysically adjacent cell types. This is highly desirable for manytissues, where cells mediating disparate functions are physicallytightly intermingled (e.g. subtypes of neurons mediating breathing,eating and pain functions in the mammalian brainstem or differentfunctional immune cell states in a malignant tumor). Furthermore,Cell-SELECT can be used for functional correction or reprogramming ofspecific cell types in both genetic as well as non-heritable diseases.

FIG. 1 shows a non-limiting exemplary schematic illustration of theCell-SELECT method for cell-type-specific expression of effector genes.The gene editing reagents (e.g., gRNA and Cas9 or other programmablenuclease) as well as the Cell-SELECT donor construct carrying theeffector gene/genes can be introduced to the cell through viral ornon-viral methods. gRNA-guided Cas9 can cleave both the target region inthe genome as well as linearize the Cell-SELECT donor construct. Thedonor construct can become incorporated to the cleaved target region inthe genome through non-homologous end joining (NHEJ) dependent DNArepair mechanism. The target regions can be in the intronic regions ofcell-type-specifically expressed genes (here Gene A is selectivelyexpressed in cell type A but not in cell type B). After the knock-in ofthe Cell-SELECT construct the effector gene can be exclusively expressedin cell type A by getting spliced to an exon of the actively transcribedGene through its splice acceptor site. There can be no expression of theeffector gene in cell type B as the targeted gene is not activelytranscribed (gRNA, guide RNA; SA, splice acceptor; SCP, self-cleavingpeptide sequence).

Emerging cellular profiling technologies have revealed a rich diversityof molecularly defined cell types in multiple organs including blood,immune system and the brain. As most disease conditions only affect oneor just a few cell types in the body, precision therapies correcting aparticular cell type have become a highly attractive avenue fortherapeutic intervention. While performing cell type specific therapiesin self renewing tissues such as the immune system has led to a plethoraof transformative treatments, achieving the same in non-regeneratingnervous system has not previously been feasible. The present disclosureprovides Cell-type control via Splice-capture of Expressed LocusEffector by CRISPR Targeting (Cell-SELECT) methods and compositions forefficient targeted knock-in in both dividing and non-dividing cells forprecise and specific functional manipulation of molecularly distinctcell types. The disclosed methods and compositions provide a means toactivate and inactivate desired cell populations without usinggenetically modified animals. The methods and compositions providedherein enable cell-type-specific therapies for human brain disorders.Recent advances in high throughput single cell technologies haverevealed a few thousand molecular cell types in the nervous system thatmediate all aspects of brain function. Importantly, the majority ofneurological disorders ranging from chronic pain and addiction tohypertension and others are the consequence of deficits in only a tinyfraction of these neuron types. A key challenge therefore inexperimental models of neurological disease as well as clinicaltranslation is to gain selective functional control over the defectivecircuit nodes and limit the corrective manipulation to the problematiccell type.

Provided herein are Cell-SELECT methods and compositions for precise andspecific functional manipulation of molecularly distinct cell types inthe brain by tapping into the transcriptional machinery that defines themolecular identity of the neuron. Disclosed herein includes an in vivogene editing approach to introduce a corrective effector gene into acell type specifically expressed genetic locus and transcribe it bysplicing it to the native transcript (FIG. 2). This approach, in someembodiments, enables a user to either selectively ablate, silence oractivate distinct neuron types expressing a given gene and consequentlyto specifically regulate the behavioral/physiological function theyexecute. An in vivo gene editing known as Homology Independent TargetedIntegration (HITI) CRISPR that allows for high fidelity (up to ˜60%)targeted genomic knock-ins of long DNA fragments in adult neural tissue.The compositions and methods provided herein allow delivery of thedisclosed Cell-SELECT constructs to any genomic locus and consequentlyfunctionally target the vast majority of cell types in the brain that todate have not been selectively targetable by transgenic organismindependent technology. Importantly, the disclosed methods andcompositions can be applied as is to non-transgenic animals and enablesclinical applications for precision in situ circuit engineering in thebrain. In some embodiments, the Cell-SELECT technology disclosed hereinis employed to gain functional control over the mammalian pain system.FIG. 2 shows a non-limiting exemplary schematic illustration of theCell-SELECT methods and compositions provided herein. The Cell Trapmethod can employ in vivo gene editing for cell-type-specific expressionof effector proteins. Cas9 as well as the Cell Trap donor construct canbe delivered with an AAV viral vector. Guide RNA can cut the targetlocus (Gene A intron) in the genome as well as the donor nucleic acid(donor insert), which can get integrated to the genome by non-homologydependent DNA repair. As Gene A is selectively expressed in Cell type“A” but not “B”, the Cell Trap effector gene (transgene) will only beexpressed in Cell Type “A” (gRNA, guide RNA; SA, splice acceptor; P2A,self-cleaving peptide sequence).

There are provided, in some embodiments, methods and compositions forincorporating an effector gene into the genome of a cell. The method cancomprise introducing into a cell: (i) a programmable nuclease or anucleic acid encoding the programmable nuclease; (ii) a targetingmolecule or a nucleic acid encoding the targeting molecule, and (iii) adonor nucleic acid or a nucleic acid encoding the donor nucleic acid,wherein the donor nucleic acid comprises a recognition site, a spliceacceptor site, a self-cleaving peptide sequence, an effector gene, andan optional transcript stabilization element. In some embodiments, thecell comprises a target gene differentially expressed in a unique celltype and/or in a cell during a unique cell state, wherein the targetgene comprises an intron comprising the recognition site. In someembodiments, the targeting molecule is complementary to the recognitionsite and the programmable nuclease is capable of cleaving therecognition site, whereby the donor nucleic acid is capable of beingincorporated into the intron through non-homologous end joining(NHEJ)-dependent DNA repair.

There are provided, in some embodiments, methods and compositions fortreating a disease or disorder in a subject. The method can compriseintroducing into a cell of a subject in need thereof: (i) a programmablenuclease or a nucleic acid encoding the programmable nuclease; (ii) atargeting molecule or a nucleic acid encoding the targeting molecule,and (iii) a donor nucleic acid or a nucleic acid encoding the donornucleic acid. In some embodiments, the donor nucleic acid comprises arecognition site, a splice acceptor site, a self-cleaving peptidesequence, an effector gene, and an optional transcript stabilizationelement. In some embodiments, the cell comprises a target genedifferentially expressed in a unique cell type and/or in a cell during aunique cell state, wherein the target gene comprises an introncomprising the recognition site. In some embodiments, the targetingmolecule is complementary to the recognition site and programmablenuclease is capable of cleaving the recognition site, whereby the donornucleic acid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair, and thereby theintroducing treats the disease or disorder in the subject.

Disclosed herein include compositions. In some embodiments, thecomposition comprises: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and/or (iii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid. In someembodiments, the donor nucleic acid comprises a recognition site, asplice acceptor site, a self-cleaving peptide sequence, an effectorgene, and an optional transcript stabilization element, and wherein thetargeting molecule is complementary to the recognition site. In someembodiments, a cell of a subject comprises a target gene differentiallyexpressed in a unique cell type and/or in a cell during a unique cellstate, wherein the target gene comprises an intron comprising therecognition site. In some embodiments, the programmable nuclease iscapable of cleaving the recognition site, whereby the donor nucleic acidis capable of being incorporated into the intron through non-homologousend joining (NHEJ)-dependent DNA repair.

There are provided, in some embodiments, methods and compositions forincorporating an effector gene into the genome of a cell. The method cancomprise introducing into a cell: (i) a programmable nuclease or anucleic acid encoding the programmable nuclease, wherein theprogrammable nuclease comprises a zinc finger nuclease (ZFN) and/ortranscription activator-like effector nuclease (TALEN); and (ii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid. In someembodiments, the donor nucleic acid comprises a recognition site, asplice acceptor site, a self-cleaving peptide sequence, an effectorgene, and an optional transcript stabilization element. In someembodiments, the cell comprises a target gene differentially expressedin a unique cell type and/or in a cell during a unique cell state,wherein the target gene comprises an intron comprising the recognitionsite. In some embodiments, the programmable nuclease is capable ofcleaving the recognition site, whereby the donor nucleic acid is capableof being incorporated into the intron through non-homologous end joining(NHEJ)-dependent DNA repair.

There are provided, in some embodiments, methods and compositions fortreating a disease or disorder in a subject. The method can compriseintroducing into a cell of a subject in need thereof: (i) a programmablenuclease or a nucleic acid encoding the programmable nuclease, whereinthe programmable nuclease comprises a zinc finger nuclease (ZFN) and/ortranscription activator-like effector nuclease (TALEN); and (ii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid. In someembodiments, the donor nucleic acid comprises a recognition site, asplice acceptor site, a self-cleaving peptide sequence, an effectorgene, and an optional transcript stabilization element. In someembodiments, the cell comprises a target gene differentially expressedin a unique cell type and/or in a cell during a unique cell state,wherein the target gene comprises an intron comprising the recognitionsite. In some embodiments, the programmable nuclease is capable ofcleaving the recognition site, whereby the donor nucleic acid is capableof being incorporated into the intron through non-homologous end joining(NHEJ)-dependent DNA repair, and thereby the introducing treats thedisease or disorder in the subject.

Disclosed herein include compositions. In some embodiments, thecomposition comprises: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease, wherein the programmable nucleasecomprises a zinc finger nuclease (ZFN) and/or transcriptionactivator-like effector nuclease (TALEN); and/or (ii) a donor nucleicacid or a nucleic acid encoding the donor nucleic acid. In someembodiments, the donor nucleic acid comprises a recognition site, asplice acceptor site, a self-cleaving peptide sequence, an effectorgene, and an optional transcript stabilization element. In someembodiments, a cell of a subject comprises a target gene differentiallyexpressed in a unique cell type and/or in a cell during a unique cellstate, wherein the target gene comprises an intron comprising therecognition site. In some embodiments, the programmable nuclease iscapable of cleaving the recognition site, whereby the donor nucleic acidis capable of being incorporated into the intron through non-homologousend joining (NHEJ)-dependent DNA repair.

In some embodiments, the method achieves an at least 10% highertargeting efficiency as compared to a homology directed repair(HDR)-based method. The targeting efficiency of the method can be, orcan be about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%,or a number or a range between any two of these values, higher than thetargeting efficiency of a homology directed repair (HDR)-based method.

In some embodiments, the method achieves an at least 10% highertargeting efficiency as compared to a Homology Independent TargetedIntegration (HITI)-based method integrating an effector gene into anexon. The targeting efficiency of the method can be, or can be about,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or a number or arange between any two of these values, higher than a HomologyIndependent Targeted Integration (HITI)-based method integrating aneffector gene into an exon.

In some embodiments, the effector gene is not expressed in a cell otherthan the unique cell type. The expression of the effector gene in a cellother than the unique cell type can be less than about 5 percent of theexpression of the effector gene in the unique cell type. The unique celltype can comprise a unique gene expression pattern. The unique cell typecan comprise a unique anatomic location. The unique cell type cancomprise anatomically locally unique gene expression.

In some embodiments, the effector gene is not expressed in a cell otherthan in a cell during the unique cell state. The expression of theeffector gene in a cell not in the unique cell state can be less thanabout 10% of the expression of the effector gene in a cell during theunique cell state. The expression of the effector gene in a cell not inthe unique cell state can be, or can be about, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%,600%, 700%, 800%, 900%, 1000%, or a number or a range between any two ofthese values, less than the expression of the effector gene in a cellduring the unique cell state. In some embodiments, the effector gene isonly expressed in a cell expressing the target gene. The expression ofthe effector gene in a cell that does not express the target gene can beless than about 5 percent of the expression of the effector gene in acell that does express the target gene. The expression of the effectorgene in a cell that does not express the target gene can be, or can beabout, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or a numberor a range between any two of these values, less than the expression ofthe effector gene in a cell that does express the target gene. Theunique cell state can be caused by hereditable, environmental, and/oridiopathic factors.

In some embodiments the target gene is a protein-encoding gene. In someembodiments the target gene is a not a protein-encoding gene. The targetgene can have one or more introns. The target gene may or may not havean intron. In some embodiments, the target gene is a long noncoding RNA(lncRNA). In some embodiments, the donor nucleic acid is integrated intothe target gene in both desired cells (e.g., a unique cell type and/or acell in a unique cell state) as well as non-desired cells (e.g., cellsother than a unique cell type or a cell in a unique cell state). In somesuch embodiments, the effector gene is expressed in the desired cells(e.g., a unique cell type and/or a cell in a unique cell state)expressing the targeted gene. In some embodiments, non-desired cells(e.g., cells other than a unique cell type or a cell in a unique cellstate) do not express the target gene, and therefore the effector genewill be dormant (e.g., not expressed).

The cell can comprise a plurality of cells. The donor nucleic acid canbe capable of being incorporated into the intron of a post-mitotic cell.The cell can be a dividing cell, a non-dividing cell, a post-mitoticcell, or any combination thereof. In some embodiments, the cell is notin G2/M phase. The cell can be a eukaryotic cell (e.g., an immune cell,an epithelial cell, a muscle cell, an endothelial cell, a neuron, a stemcell, or any combination thereof). The immune cell can comprise a Tcell, a B cell, a natural killer cell, a monocyte, a macrophage cell, adendritic cell, or any combination thereof. The stem cell can comprisean embryonic stem cell, an induced pluripotent stem cell (iPSC), ahematopoietic stem/progenitor cell (HSPC), or any combination thereof.The cell can be a germline cell or a somatic cell. Exemplary cellscontemplated as described herein, include, a bacterial cell; an archaealcell; a single-celled eukaryotic organism; a plant cell; an algal cell;a fungal cell; an animal cell; a cell from an invertebrate animal (e.g.,an insect, a cnidarian, an echinoderm, a nematode, etc.); a eukaryoticparasite (e.g., a malarial parasite, e.g., Plasmodium falciparum; ahelminth; etc.); a cell from a vertebrate animal (e.g., fish, amphibian,reptile, bird, mammal); a mammalian cell, e.g., a rodent cell, a humancell, a non-human primate cell. Suitable host cells include naturallyoccurring cells; genetically modified cells (e.g., cells geneticallymodified in a laboratory, e.g., by the “hand of man”); and cellsmanipulated in vitro in any way.

Effector Genes

The cell type specific functional manipulations disclosed herein candepend on the effector gene carried by the Cell-SELECT donor construct(e.g., donor nucleic acid). The effector genes that can be introduced tothe cell through the Cell-SELECT method include, but are not limited to,fluorescent or other contrast generating protein encoding genes (e.g.Gfp, mCherry), cell apoptosis triggering genes (e.g. Casp3), particularcell-type function regulating genes (e.g. ion channels, GPCR-s, enzymesetc.) or any other recombinant protein. The expression of the effectorgene can be capable of modulating cellular membrane potential. Theexpression of the effector gene can be capable of altering the membranepotential of the cell by depolarizing the cell and/or hyperpolarizingthe cell. The expression of the effector gene can be capable of reducingsynaptic transmission by at least 10 percent. The expression of theeffector gene can be capable of blocking synaptic transmission. Theeffector gene can comprise Kir2.1. The expression of the effector genecan be capable of blocking synaptic transmission. The effector gene cancomprise tetanus toxin (TNT). The expression of the effector gene can becapable of sensitizing the cell to a pharmacological compound,temperature change, or light. The effector gene can comprise a DREADDreceptor and/or Channelrhodopsin-2. The effector gene can be capable ofsensitizing the cell to a drug and/or to a prodrug. The effector genecan comprise cytosine deaminase and/or uracil phosphoribosyl transferaseand the prodrug can comprise 5-fluorocytosine (5-FC). The effector genecan comprise thymidine kinase (TK), and wherein the prodrug can compriseganciclovir. The effector gene can comprise an enzyme, a signaltransduction protein, an ion-channel and/or a G-protein coupled receptor(GPCR). In some embodiments, the effector gene encodes a recombinantprotein and/or a native protein. The effector gene can be capable ofinducing cell death. The effector gene can comprise cytosine deaminase,thymidine kinase, Bax, Bid, Bad, Bak, BCL2L11, p53, PUMA, Diablo/SMAC,S-TRAIL, Cas9, Cas9n, hSpCas9, hSpCas9n, HSVtk, cholera toxin,diphtheria toxin, alpha toxin, anthrax toxin, exotoxin, pertussis toxin,Shiga toxin, shiga-like toxin Fas, TNF, caspase 2, caspase 3, caspase 6,caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12,purine nucleoside phosphorylase, or any combination thereof. The uniquecell state can comprise a senescent cell state induced by a tumormicroenvironment. The senescent cell state induced by a tumormicroenvironment can comprise expression of CD57, KRLG1, TIGIT, or anycombination thereof. The effector gene can comprise interleukin-12(IL-12).

Although chronic control of cellular function can be easiest to achieve,temporally controlled regulation of neuronal output is often desirablefor meaningful neural circuit control. There are provided, in someembodiments, Cell-SELECT compositions and methods, including constructs,to deliver genes that make the cells sensitive to novel pharmacologicalcompounds (using DREADD receptors) or light (implementingChannelrhodopsin 2) to the pain processing neurons in LPBN. There isprovided, in some embodiments, a pAAV-CELL-SELECT construct for targetedviral knock-in of optogenetic activatory channelrhodopsin-2 (SEQ ID NO:3). The effector gene can comprise a H134R mutant of an N-terminalfragment of channelrhodopsin-2 from the alga C. reinhardtii. Thedisclosed methods and compositions can enable pharmacological and/orlight control over pain behaviors conditional on external delivery ofcontrol agents. In some embodiments, response decay times as wellpotential habituation effects can be effectively managed for theprovided methods. The Cell-SELECT methods can enable precise regulationof local brain function (e.g., gain functional control over cell typesin the brain). The disclosed compositions and methods can be modular andeasily applicable to control any molecularly distinct neuron type in anyneural circuit and importantly also applicable to a clinical setting.

There are provided, in some embodiments, methods and compositions forfunctional reprogramming of disease-associated cell states in the immunesystem. The disclosed Cell-SELECT method can be used to functionallyreprogram specific cell states in non-genetic diseases that stem fromenvironmental and/or idiopathic factors. For example, T-lymphocytes thathave migrated to a malignant tumor can sometimes become senescent andfail to mediate an antitumor response. This senescent cell state can becharacterized by the expression of known genetic programs (e.g.expression of CD57, KRLG1, and other genes). This senescent state can beovercome by exposure to cytokines such as interleukin-12 (IL12). ACell-SELECT-based approach provided herein can be deployed toselectively rescue T-lymphocytes from the senescence by deliveringCell-SELECT donors containing the IL12 gene coding sequence with Cas9and KRLG1 gene intron targeting gRNA with AAV6 based viral vectors inthe tumor environment. This can result in genomic knock-in of IL12coding sequence to many disparate cell types in the tumormicroenvironment. However, IL12 can be selectively be expressed andreleased from senescent T-lymphocytes that enable them to reactivatetheir anti-tumor response via autocrine signaling.

In some embodiments, the effector gene encodes a protein of interest. Asused herein, a “protein of interest” can be any protein, includingnaturally-occurring and non-naturally occurring proteins. Examples ofprotein of interest include, but are not limited to, luciferases;fluorescent proteins (e.g., GFP); growth hormones (GHs) and variantsthereof; insulin-like growth factors (IGFs) and variants thereof;granulocyte colony-stimulating factors (G-CSFs) and variants thereof;erythropoietin (EPO) and variants thereof; insulin, such as proinsulin,preproinsulin, insulin, insulin analogs, and the like; antibodies andvariants thereof, such as hybrid antibodies, chimeric antibodies,humanized antibodies, monoclonal antibodies; antigen binding fragmentsof an antibody (Fab fragments), single-chain variable fragments of anantibody (scFV fragments); dystrophin and variants thereof; clottingfactors and variants thereof; cystic fibrosis transmembrane conductanceregulator (CFTR) and variants thereof; and interferons and variantsthereof.

In some embodiments, the protein of interest is a therapeutic protein orvariant thereof. Non-limiting examples of therapeutic proteins includeblood factors, such as β-globin, hemoglobin, tissue plasminogenactivator, and coagulation factors; colony stimulating factors (CSF);interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF),stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGFand acidic FGF), hepatocyte growth factor (HGF), insulin-like growthfactors (IGFs), bone morphogenetic protein (BMP), epidermal growthfactor (EGF), growth differentiation factor-9 (GDF-9), hepatoma derivedgrowth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF),neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin(TPO), transforming growth factor alpha (TGF-a), transforming growthfactor beta (TGF-β), and the like; soluble receptors, such as solubleTNF-receptors, soluble VEGF receptors, soluble interleukin receptors(e.g., soluble IL-1 receptors and soluble type II IL-1 receptors),soluble γ/δ T cell receptors, ligand-binding fragments of a solublereceptor, and the like; enzymes, such as -glucosidase, imiglucarase,β-glucocerebrosidase, and alglucerase; enzyme activators, such as tissueplasminogen activator; chemokines, such as IP-10, monokine induced byinterferon-gamma (Mig), Gro/IL-8, RANTES, MIP-1, MIP-I β, MCP-1, PF-4,and the like; angiogenic agents, such as vascular endothelial growthfactors (VEGFs, e.g., VEGF121 , VEGF165, VEGF-C, VEGF-2), transforminggrowth factor-beta, basic fibroblast growth factor, glioma-derivedgrowth factor, angiogenin, angiogenin-2; and the like; anti-angiogenicagents, such as a soluble VEGF receptor; protein vaccine; neuroactivepeptides, such as nerve growth factor (NGF), bradykinin,cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasinghormone, beta-endorphin, enkephalin, substance P, somatostatin,prolactin, galanin, growth hormone-releasing hormone, bombesin,dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y,luteinizing hormone, calcitonin, insulin, glucagons, vasopressin,angiotensin II, thyrotropin-releasing hormone, vasoactive intestinalpeptide, a sleep peptide, and the like; thrombolytic agents; atrialnatriuretic peptide; relaxin; glial fibrillary acidic protein; folliclestimulating hormone (FSH); human alpha-1 antitrypsin; leukemiainhibitory factor (LIF); transforming growth factors (TGFs); tissuefactors, luteinizing hormone; macrophage activating factors; tumornecrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growthfactor; tissue inhibitors of metalloproteinases; vasoactive intestinalpeptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptorantagonists; and the like. Some other non-limiting examples of proteinof interest include ciliary neurotrophic factor (CNTF); brain-derivedneurotrophic factor (BDNF); neurotrophins 3 and 4/5 (NT-3 and 4/5);glial cell derived neurotrophic factor (GDNF); aromatic amino aciddecarboxylase (AADC); hemophilia related clotting proteins, such asFactor VIII, Factor IX, Factor X; dystrophin or mini-dystrophin;lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storagedisease-related enzymes, such as glucose-6-phosphatase, acid maltase,glycogen debranching enzyme, muscle glycogen phosphorylase, liverglycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase(e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, β-enolase,and glycogen synthase; lysosomal enzymes (e.g.,beta-N-acetylhexosaminidase A); and any variants thereof.

In some embodiments, the protein of interest is an active fragment of aprotein, such as any of the proteins disclosed herein. In someembodiments, the protein of interest is a fusion protein comprising someor all of two or more proteins. In some embodiments a fusion protein cancomprise all or a portion of any of the aforementioned proteins.

In some embodiments, the method is multiplexed. In some embodiments twoor more genetic loci (e.g., introns) are targeted for knock-in. In somesuch embodiments, all of the target genes are expressed in a unique celltype and/or unique cell state. For example, the method can compriseintegrating a first donor nucleic acid (comprising a first effectorgene) into an intron of a first target gene and integrating a seconddonor nucleic acid (comprising a second effector gene) into an intron ofa second target gene. The first target gene and the second target genecan be expressed in the same or different unique cell type and/or uniquecell state. The first effector gene and the second effector gene can bethe same or different. The first effector gene and the second effectorgene can encode for two or more proteins of interest. The effector genecan comprise coding regions for two or more proteins of interest. Thetwo or more proteins of interest can be the same or different from eachother. In some embodiments, the two or more proteins of interest arerelated polypeptides, for example neutralizing antibodies for the samevirus.

In some embodiments, the protein of interest is a multi-subunit protein.For examples, the protein of interest can comprise two or more subunits,or two or more independent polypeptide chains. In some embodiments, theprotein of interest can be an antibody. Examples of antibodies include,but are not limited to, antibodies of various isotypes (for example,IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM); monoclonal antibodiesproduced by any means known to those skilled in the art, including anantigen-binding fragment of a monoclonal antibody; humanized antibodies;chimeric antibodies; single-chain antibodies; antibody fragments such asFv, F(ab′)2, Fab′, Fab, Facb, scFv and the like; provided that theantibody is capable of binding to antigen. In some embodiments, theantibody is a full-length antibody.

In some embodiments, the effector gene encodes a pro-survival protein(e.g., Bcl-2, Bcl-XL, Mcl-1 and A1). In some embodiments, the effectorgene encodes a apoptotic factor or apoptosis-related protein such as,for example, AIF, Apaf e.g. Apaf-1, Apaf-2, Apaf-3, oder APO-2 (L),APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x_(L), Bcl-xs, bik, CAD,Calpain, Caspase e.g. Caspase-1, Caspase-2, Caspase-3, Caspase-4,Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10,Caspase-11, ced-3, ced-9, c-Jun, c-Myc, crm A, cytochrom C, CdR1, DcR1,DD, DED, DISC, DNA-PKcs, DR3, DR4, DRS, FADD/MORT-1, FAK, Fas(Fas-ligand CD95/fas (receptor)), FLICE/MACH, FLIP, fodrin, fos,G-Actin, Gas-2, gelsolin, granzyme A/B, ICAD, ICE, JNK, Lamin A/B, MAP,MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD, NF-_(kappa)B, NuMa, p53, PAK-2,PARP, perforin, PITSLRE, PKCdelta, pRb, presenilin, prICE, RAIDD, Ras,RIP, sphingomyelinase, thymidinkinase from herpes simplex, TRADD, TRAF2,TRAIL-R1, TRAIL-R2, TRAIL-R3, and/or transglutaminase.

In some embodiments, the effector gene encodes a cellular reprogrammingfactor capable of converting an at least partially differentiated cellto a less differentiated cell, such as, for example, Oct-3, Oct-4, Sox2,c-Myc, Klf4, Nanog, Lin28, ASCL1, MYT1 L, TBX3b, SV40 large T, hTERT,miR-291, miR-294, miR-295, or any combinations thereof. In someembodiments, the effector gene encodes a programming factor that iscapable of differentiating a given cell into a desired differentiatedstate, such as, for example, nerve growth factor (NGF), fibroblastgrowth factor (FGF), interleukin-6 (IL-6), bone morphogenic protein(BMP), neurogenin3 (Ngn3), pancreatic and duodenal homeobox 1 (Pdx1),Mafa, or any combination thereof.

In some embodiments, the effector gene encodes a human adjuvant proteincapable of eliciting an innate immune response, such as, for example,cytokines which induce or enhance an innate immune response, includingIL-2, IL-12, IL-15, IL-18, IL-21CCL21, GM-CSF and TNF-alpha; cytokineswhich are released from macrophages, including IL-1, IL-6, IL-8, IL-12and TNF-alpha; from components of the complement system including Clq,MBL, Clr, Cls, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b,C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H, I, Pand CD59; from proteins which are components of the signaling networksof the pattern recognition receptors including TLR and IL-1 R1, whereasthe components are ligands of the pattern recognition receptorsincluding IL-1 alpha, IL-1 beta, Beta-defensin, heat shock proteins,such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen,Typ111 repeat extra domain A of fibronectin; the receptors, includingIL-1 RI, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11; the signal transducers including components of the Small-GTPasessignaling (RhoA, Ras, Rac1, Cdc42 etc.), components of the PIP signaling(PI3K, Src-Kinases, etc.), components of the MyD88-dependent signaling(MyD88, IRAK1, IRAK2, etc.), components of the MyD88-independentsignaling (TICAM1, TICAM2, etc.); activated transcription factorsincluding e.g. NF-_(κ)B, c-Fos, c-Jun, c-Myc; and induced target genesincluding e.g. IL-1 alpha, IL-1 beta, Beta-Defensin, IL-6, IFN gamma,IFN alpha and IFN beta; from costimulatory molecules, including CD28 orCD40-ligand or PD1; protein domains, including LAMP; cell surfaceproteins; or human adjuvant proteins including CD80, CD81, CD86, trif,flt-3 ligand, thymopentin, Gp96 or fibronectin, or any species homologof any of the above human adjuvant proteins.

In some embodiments, the effector gene encodes immunogenic materialcapable of stimulating an immune response (e.g., an adaptive immuneresponse) such as, for example, antigenic peptides or proteins from apathogen. The expression of the antigen may stimulate the body'sadaptive immune system to provide an adaptive immune response. Thus, itis contemplated that some embodiments the Cell-SELECT compositionsprovided herein can be employed as vaccines for the prophylaxis ortreatment of infectious diseases (e.g., as vaccines).

The effector gene can comprise a suicide gene. In some embodiments, theeffector gene encodes a protein which, upon administration of a prodrug,causes the death of its host cell (e.g., a suicide gene). In someembodiments, the methods herein comprise a) administering (i) aprogrammable nuclease or a nucleic acid encoding the programmablenuclease; (ii) a targeting molecule or a nucleic acid encoding thetargeting molecule, and/or (iii) a donor nucleic acid or a nucleic acidencoding the donor nucleic acid to a subject comprising the cell and b)administering a prodrug to the subject. Any suitable suicide gene andprodrug is contemplated this disclosure, such as, for example, thesuicide gene/prodrug combinations depicted in Table 1.

TABLE 1 EFFECTOR GENES AND PRODRUGS Effector (suicide) Gene Prodrug HSVthymidine Ganciclovir (GCV); Ganciclovir elaidic acid kinase (TK) ester;Penciclovir (PCV); Acyclovir (ACV); Valacyclovir (VCV);(E)-5-(2-bromovinyl)- 2′-deoxyuridine (BVDU); Zidovuline (AZT);2′-exo-methanocarbathymidine (MCT) Cytosine Deaminase 5-fluorocytosine(5-FC) (CD) Purine nucleoside 6-methylpurine deoxyriboside (MEP);phosphorylase (PNP) fludarabine (FAMP) Cytochrome p450 Cyclophosphamide(CPA); Ifosfamide (IFO); enzymes (CYP) 4-ipomeanol (4-IM)Carboxypeptidases 4-[(2-chloroethyl)(2- (CP)mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA); Hydroxy-andamino-aniline mustards; Anthracycline glutamates; Methotrexate α-peptides (MTX-Phe) Caspase-9 AP1903 Carboxylesterase Irinotecan (IRT);Anthracycline acetals (CE) Nitroreductase dinitroaziridinylbenzamideCB1954; (NTR) dinitrobenzamide mustard SN23862; 4- Nitrobenzylcarbamates; Quinones Horse radish Indole-3-acetic acid (IAA);5-Fluoroindole- peroxidase (HRP) 3-acetic acid (FIAA) Guanine6-Thioxanthine (6-TX) Ribosyltransferase (XGRTP) Glycosidase enzymesHM1826; Anthracycline acetals Methionine-α,γ- Selenomethionine (SeMET)lyase (MET) Thymidine 5′-Deoxy-5-fluorouridine (5′-DFU) phosphorylase(TP)

A protein of interest encoded by an effector gene can be of variouslengths. For example, the protein of interest can be at least about 200amino acids, at least about 250 amino acids, at least about 300 aminoacids, at least about 350 amino acids, at least about 400 amino acids,at least about 450 amino acids, at least about 500 amino acids, at leastabout 550 amino acids, at least about 600 amino acids, at least about650 amino acids, at least about 700 amino acids, at least about 750amino acids, at least about 800 amino acids, or longer in length. Insome embodiments, the protein of interest is at least about 480 aminoacids in length. In some embodiments, the protein of interest is atleast about 500 amino acids in length. In some embodiments, the proteinof interest is about 750 amino acids in length.

A donor nucleic acid can comprise one effector gene, or more than oneeffector gene. The effector genes can have different lengths indifferent implementations. In some embodiments, an effector genes is, oris about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250,3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500,8000, 8500, 9000, 9500, 10000, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, an effectorgene is at least, or is at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500,510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000 nucleotides inlength.

Methods of Diagnosing and Treating Diseases and Disorders

Disclosed herein include methods of treating a disease or disorder. Insome embodiments, the unique cell type and/or the cell in the uniquecell state causes and/or aggravates a disease or disorder. The uniquecell type and/or the cell in the unique cell state can be associatedwith the pathology of a disease or disorder. The cell can be the cell ofa subject. The cell can be the cell of a subject suffering from adisease or disorder. The disease or disorder can be a blood disease, animmune disease, a cancer, an infectious disease, a genetic disease, adisorder caused by aberrant mtDNA, a metabolic disease, a disordercaused by aberrant cell cycle, a disorder caused by aberrantangiogenesis, a disorder cause by aberrant DNA damage repair, or anycombination thereof. In some embodiments, the unique cell type and/orthe cell in the unique cell state causes and/or aggravates a disease ordisorder. The unique cell type and/or the cell in the unique cell statecan be associated with the pathology of a disease or disorder.

The disease or disorder can comprise a neurological disease or disorder.The cell, the unique cell type and/or the cell in the unique cell statecan comprise a neuron. The cell, the unique cell type and/or the cell inthe unique cell state can comprise a cell in a brain region. The neuroncan be involved in memory expression, involved in eating control,involved in addiction, a component of a motor control circuit, ananxiety processing neuron, an analgesia inducing neuron, an analgesiaprocessing neuron, a pain-processing neuron, or any combination thereof.The neuron can be associated with a neurological disease or disorder. Insome embodiments, the neuron can compensate, reverse or alleviate adisorder/disease state. The neurological disease or disorder cancomprise Alzheimer's disease, Creutzfeld-Jakob's syndrome/disease,bovine spongiform encephalopathy (BSE), prion related infections,diseases involving mitochondrial dysfunction, diseases involvingβ-amyloid and/or tauopathy, Down's syndrome, hepatic encephalopathy,Huntington's disease, motor neuron diseases, amyotrophic lateralsclerosis (ALS), olivoponto-cerebellar atrophy, post-operative cognitivedeficit (POCD), systemic lupus erythematosus, systemic clerosis,Sjogren's syndrome, Neuronal Ceroid Lipofuscinosis, neurodegenerativecerebellar ataxias, Parkinson's disease, Parkinson's dementia, mildcognitive impairment, cognitive deficits in various forms of mildcognitive impairment, cognitive deficits in various forms of dementia,dementia pugilistica, vascular and frontal lobe dementia, cognitiveimpairment, learning impairment, eye injuries, eye diseases, eyedisorders, glaucoma, retinopathy, macular degeneration, head or brain orspinal cord injuries, head or brain or spinal cord trauma, convulsions,epileptic convulsions, epilepsy, temporal lobe epilepsy, myoclonicepilepsy, tinnitus, dyskinesias, chorea, Huntington's chorea, athetosis,dystonia, stereotypy, ballism, tardive dyskinesias, tic disorder,torticollis spasmodicus, blepharospasm, focal and generalized dystonia,nystagmus, hereditary cerebellar ataxias, corticobasal degeneration,tremor, essential tremor, addiction, anxiety disorders, panic disorders,social anxiety disorder (SAD), attention deficit hyperactivity disorder(ADHD), attention deficit syndrome (ADS), restless leg syndrome (RLS),hyperactivity in children, autism, dementia, dementia in Alzheimer'sdisease, dementia in Korsakoff syndrome, Korsakoff syndrome, vasculardementia, dementia related to HIV infections, HIV-1 encephalopathy, AIDSencephalopathy, AIDS dementia complex, AIDS-related dementia, majordepressive disorder, major depression, depression, memory loss, stress,bipolar manic-depressive disorder, drug tolerance, drug tolerance toopioids, movement disorders, fragile-X syndrome, irritable bowelsyndrome (IBS), migraine, multiple sclerosis (MS), muscle spasms, pain,chronic pain, acute pain, inflammatory pain, neuropathic pain,posttraumatic stress disorder (PTSD), schizophrenia, spasticity,Tourette's syndrome, eating disorders, food addiction, binge eatingdisorders, agoraphobia, generalized anxiety disorder,obsessive-compulsive disorder, panic disorder, social phobia, phobicdisorders, substance-induced anxiety disorder, delusional disorder,schizoaffective disorder, schizophreniform disorder, substance-inducedpsychotic disorder, hypertension, or any combination thereof. Theneurological disease or disorder can comprise a neuro-psychiatricdisorder.

The neurological disease or disorder can comprise pain (e.g., acute painand/or chronic pain). The pain can be selected from the group comprisingneuropathic pain, allodynia, hyperalgesia, dysesthesia, causalgia,neuralgia, and arthralgia. The pain can be associated with cancer, tumorpressure, bone metastasis, chemotherapy peripheral neuropathy, sciaticaradiculopathy, lumbar radiculopathy, cervical radiculopathy, failed backsurgery syndrome, piriformis syndrome, phantom pain, arachnoiditis,fibromyalgia, facet joint mediated pain, sympathetically-mediated painsyndrome, complex regional pain syndromes (crps), sacroiliac (si) jointmediated pain, meralgia paresthetica, localized myofacial painsyndromes, myofacial trigger points, diffuse myofacial pain syndrome,post-herpetic neuralgia, trigeminal neuralgia, glossopharyngealneuralgia, post-epesiotomy scar pain, post-hernia repair scar pain,post-surgery scar pain, post-radiotherapy scar pain, vulvodynia,vaginismus, levator ani syndrome, chronic prostatitis, interstitialcystitis, first bite syndrome, rheumatoid arthritis pain, osteoarthritispain, atypical odontalgia, phantom tooth pain, neuropathic orofacialpain, primary erythermalgia, atypical facial pain, or any combinationthereof.

The compositions, systems, and methods described herein can be used toexpress one or more effector genes in a unique cell type and/or in acell during a unique cell state to treat, prevent, and/or diagnosevarious diseases or disorders. Non-limiting examples of the diseasesinclude cancer such as carcinoma, sarcoma, leukemia, lymphoma; andautoimmune diseases such as multiple sclerosis. Non-limiting examples ofcarcinomas include esophageal carcinoma; hepatocellular carcinoma; basalcell carcinoma, squamous cell carcinoma (various tissues); bladdercarcinoma, including transitional cell carcinoma; bronchogeniccarcinoma; colon carcinoma; colorectal carcinoma; gastric carcinoma;lung carcinoma, including small cell carcinoma and non-small cellcarcinoma of the lung; adrenocortical carcinoma; thyroid carcinoma;pancreatic carcinoma; breast carcinoma; ovarian carcinoma; prostatecarcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous glandcarcinoma; papillary carcinoma; papillary adenocarcinoma;cystadenocarcinoma; medullary carcinoma; renal cell carcinoma; ductalcarcinoma in situ or bile duct carcinoma; choriocarcinoma; seminoma;embryonal carcinoma; Wilm's tumor; cervical carcinoma; uterinecarcinoma; testicular carcinoma; osteogenic carcinoma; epitheliealcarcinoma; and nasopharyngeal carcinoma. Non-limiting examples ofsarcomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,chordoma, osteogenic sarcoma, osteosarcoma, angio sarcoma, endotheliosarcoma, lymphangio sarcoma, lymphangioendothelio sarcoma, synovioma,mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, andother soft tissue sarcomas. Non-limiting examples of solid tumorsinclude glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma. Non-limiting examples of leukemias include chronicmyeloproliferative syndromes; acute myelogenous leukemias; chroniclymphocytic leukemias, including B-cell CLL, T-cell CLL prolymphocyteleukemia, and hairy cell leukemia; and acute lymphoblastic leukemias.Examples of lymphomas include, but are not limited to, B-cell lymphomas,such as Burkitt's lymphoma; Hodgkin's lymphoma; and the like. Othernon-liming examples of the diseases that can be treated using theCell-SELECT methods and compositions disclosed herein include geneticdisorders including sickle cell anemia, cystic fibrosis, lysosomal acidlipase (LAL) deficiency 1, Tay-Sachs disease, Phenylketonuria,Mucopolysaccharidoses, Glycogen storage diseases (GSD, e.g., GSD typesI, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and XIV),Galactosemia, muscular dystrophy (e.g., Duchenne muscular dystrophy),and hemophilia.

Additionally, the Cell-SELECT compositions, systems, and methodsprovided herein may be used for the diagnosis, treatment, and/orprevention of infectious diseases. As used herein, the term “infectiousdiseases” refers to diseases caused by any pathogen or agent thatinfects mammalian cells, preferably human cells and causes a diseasecondition, such as, for example, bacteria, yeast, fungi, protozoans,mycoplasma, viruses, prions, and parasites. Non-limiting examples ofinfectious diseases include (a) viral diseases such as, for example,diseases resulting from infection by an adenovirus, a herpesvirus (e.g.,HSV-i, HSV-II, CMV, or VZV), a poxvirus (e-g˜, an orthopoxvirus such asvariola or vaccinia, or molluscum contagiosum), a picornavirus (e.g.,rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), aparamyxovirus (e.g., parainfluenza virus, mumps virus, measles vims, andrespiratory syncytial virus (RSV)), a coronavirus (e.g., SARS,SARS-Cov-2), a papovavirus (e.g., papillomaviruses, such as those thatcause genital warts, common warts, or plantar warts), a hepadnavirus(e.g., hepatitis B vims), a flavivirus (e.g., hepatitis C virus orDengue virus), or a retrovirus (e.g., a lentivirus such as HIV); (b)bacterial diseases such as, for example, diseases resulting frominfection by bacteria of, for example, the genus Escherichia,Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria,Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas,Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria,Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter,Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia,Haemophilus, or Bordeteila; (c) other infectious diseases, suchchlamydia, fungal diseases including but not limited to candidiasis,aspergillosis, histoplasmosis, cryptococcal meningitis, parasiticdiseases including but not limited to malaria, Pneumocystis carniipneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, andtrypanosome infection and prions that cause human disease such asCreutzfeldt-Jakob Disease (CJD), variant Creutzfeldt-Jakob Disease(vCJD), Gerstmann-Straussler-Scheinker syndrome, Fatal Familial Insomniaand kuru.

Diagnostic Applications

In some embodiments, the methods and compositions provided herein areuseful in detecting a disease or disorder and/or monitoring theprogression of a disease or disorder. As used herein, the term“diagnostic” refers identifying the presence or absence of or nature ofa disease or disorder. Such detection methods can be used, for example,for early diagnosis of the condition, to determine whether a subject ispredisposed to a disease or disorder, to monitor the progress of thedisease or disorder or the progress of treatment protocols, to assessthe severity of the disease or disorder, to forecast the an outcome of adisease or disorder and/or prospects of recovery, or to aid in thedetermination of a suitable treatment for a subject. The detection canoccur in vitro or in vivo. The effector gene can comprise a diagnosticagent. The effector gene can comprise a diagnostic contrast agent.

In some embodiments, the effector gene encodes a diagnostic agent. Insome embodiments, the diagnostic agent aids in the identification of aunique cell type and/or a unique cell state. The diagnostic agent can bea molecule capable of detection, including, but not limited to,fluorescers, chemiluminescers, chromophores, bioluminescent proteins,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,isotopic labels, semiconductor nanoparticles, dyes, metal ions, metalsols, ligands (e.g., biotin, streptavidin or haptens) and the like. Theterm “fluorescer” refers to a substance or a portion thereof which iscapable of exhibiting fluorescence in the detectable range. For example,the diagnostic agent may comprise, in some embodiments, a fluorescentprotein, such as, but not limited to, green fluorescent protein (GFP),enhanced green fluorescent protein (EGFP), yellow fluorescent protein(YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescentprotein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron,mApple, mCherry, rsCherry, rsCherryRev, or any combination thereof. Insome embodiments, the expression, stability, and/or activity (e.g.,fluorescence) of the diagnostic agent is configured to be responsive toa disease state or a disorder state.

In some embodiments, the diagnostic agent aids in the identification ofa unique cell type and/or a unique cell state. The unique cell typeand/or a unique cell state can comprise lesions (e.g. tumors, infectedcells). Detection and/or imaging of the diagnostic agent can enable aclinician to intraoperatively, laparoscopically, intravascularly orendoscopically detect said lesions. In some such embodiments,discrimination between lesions (e.g. tumors) and non-lesions (e.g.non-tumor tissue) is enhanced by the detection and/or imaging of thediagnostic agent. In some embodiments, detection and/or imaging of thediagnostic agent can enable a clinician to accurately locate lesions ina patient and thereby aid resection, irradiation, biopsy and/or lesionremoval. In some embodiments, detection and/or imaging of the diagnosticagent aids the detection of non-malignant pathological lesions, such as,an infarct, including myocardial, atherosclerotic plaque, clot,including thrombosis, pulmonary embolism, infectious or inflammatorylesion, non-tumorous or noninfectious inflammation, or hyperplasia. Thedetection and/or imaging of the diagnostic agent may also be used todetect various stages of progression or severity of disease (e.g.,benign, premalignant, and malignant breast lesions, tumor growth, ormetastasis). The detection and/or imaging of the diagnostic agent mayalso be used to detect the response of the disease to prophylactic ortherapeutic treatments or other interventions. The detection and/orimaging of the diagnostic agent can furthermore be used to help themedical practitioner in determining prognosis (e.g., worsening,status-quo, partial recovery, or complete recovery) of the patient, andthe appropriate course of action.

Detection and/or imaging of the diagnostic agent can be performed, forexample, using an ultrasound scanner, a magnetic resonance imaginginstrument (MRI scanner), an X-ray source with film or a detector (e.g.,conventional or digital radiography system), an X-ray computedtomography (CT) or computed axial tomography (CAT) scanner, a gammacamera, or a positron emission tomography (PET) scanner. Various medicalimaging systems have been developed for open surgery as well as forlaparoscopic, thoracoscopic, and robot-assisted surgery and can be usedin the practice of the invention. Conventional laparoscopes andendoscopes can be equipped with a photodetector (e.g., camera or CCDdetector) to provide guidance during medical procedures. Fiber-opticimaging systems can also be used, which include portable handheldmicroscopes, flexible endoscopes, and microendoscopes. For example, anillumination source can be added to such devices to allow fluorescenceimaging. A miniaturized ultrasound transducer can be added to the tip ofa laparoscope or catheter for intravascular ultrasound (IVUS) imaging.Miniaturized imaging systems can be used that allow imaging inside smallcavities and constricted spaces. In addition, miniaturized imagingdevices (e.g., microendoscopes) may be implanted within a subject forlong-term imaging studies. In addition, a camera may be used to takeboth photographic images of a subject and to detect signals from thediagnostic agent, so that photographic images of the subject and imagesof the signals from the diagnostic agent can be superimposed to allowregions containing the diagnostic agent to be mapped to the subject'sanatomy.

Donor Nucleic Acids

There are provided, in some embodiments, donor nucleic acids. The donornucleic acid can comprise one or more of a recognition site, a spliceacceptor site, a self-cleaving peptide sequence, an effector gene, and atranscript stabilization element. The self-cleaving peptide sequence cancomprise porcine teschovirus-1 2A peptide (P2A), Thosea asigna virus 2Apeptide (T2A), equine rhinitis A virus 2A peptide (E2A), foot-and-mouthdisease virus 2A peptide (F2A), or any combination thereof. Thetranscript stabilization element can be capable of enhancing thestability of a transcript of the effector gene. The transcriptstabilization element can comprise woodchuck hepatitispost-translational regulatory element (WPRE), bovine growth hormonepolyadenylation (bGH-polyA) signal sequence, human growth hormonepolyadenylation (hGH-polyA) signal sequence, or any combination thereof.The splice acceptor site can be capable of being recognized and cleavedby a spliceosome. The splice acceptor site can comprise a branchpoint, apolypyrimidine tract, a 3′ splice site, or any combination thereof. Thetranslation frame linker can ensure that the effector gene is expressedin frame with the preceding exon of the target gene. The translationframe linker can be, can be about, can be at least, or can be at most,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or anumber or a range between any of these values, of nucleotides in length.In some embodiments, the translation frame linker places the effectorgene in translational frame with the preceding exon of the target gene.The recognition site can be adjacent to a protospacer adjacent motif(PAM) capable of being recognized by the programmable nuclease. Thedonor nucleic acid can comprise a translation frame linker. The donornucleic acid can comprise the structure 5′-[recognition site]-[spliceacceptor site]-[translation frame linker]-[self-cleaving peptidesequence]-[effector gene]-3′. The donor nucleic acid can comprise thestructure 5′-[recognition site]-[splice acceptor site]-[translationframe linker]-[self-cleaving peptide sequence]-[effectorgene]-[transcript stabilization element]-3′. The donor nucleic acid cancomprise the target gene coding sequence downstream of the intron. Thedonor nucleic acid can comprise the structure 5′-[recognitionsite]-[splice acceptor site]-[target gene coding sequence downstream ofthe intron]-[self-cleaving peptide sequence]-[effector gene]-3′. Thedonor nucleic acid can comprise the structure 5′-[recognitionsite]-[splice acceptor site]-[target gene coding sequence downstream ofthe intron]-[self-cleaving peptide sequence]-[transcript stabilizationelement]-3′. The recognition site of the donor nucleic acid and therecognition site of the intron can be the same. In some embodiments, therecognition site does not exist after the donor nucleic acid has beenproperly incorporated into the intron. In some embodiments, the donornucleic acid does not comprise a promoter. In some embodiments, theintegration of the donor nucleic acid into the target gene through NHEJcan initially result in integration in the wrong orientation. In somesuch embodiments, the integration in the wrong orientation willreconstitute the recognition site in the genome and the wronglyintegrated donor nucleic acid will be cut out again. In someembodiments, only the integration in the correct orientation will breakthe original recognition site and allow the donor nucleic acid to remainin the genome. In some embodiments, the donor nucleic acid does notcomprise transcript stabilization element. In some embodiments, thedonor nucleic acid encodes a degron. In some embodiments, the degron is5′ of the self-cleaving peptide. In some embodiments, the degronprevents accumulation of an incomplete target gene product. The degroncan comprise a DHFR degron, an N-degron, a phospho degron, a heatinducible degron, a photosensitive degron, an oxygen dependent degron,ornithine decarboxylase degron, estrogen receptor domain degrons, aecDHFR degron, an FKBP degron, a UnaG degron, or any combinationthereof. The donor nucleic acid can comprise an IRES. In someembodiments, the Cell-SELECT donor can further comprise gRNA expressionconstructs, multiple gRNA cutting sites and/or other componentsfacilitating efficient cellular transduction of the Cell-SELECTreagents.

FIGS. 3A-3B show non-limiting exemplary schematic illustrations ofCell-SELECT donor constructs (e.g., donor nucleic acids) for cell typespecific expression of effector genes with their respective structuralelements. FIG. 3A shows the basic design of a CELL-SELECT donor (e.g.,donor nucleic acid) comprising the following elements: gRNA cuttingsite—for linearizing the donor construct that primes it for genomicintegration through NHEJ; splice acceptor site—ensures splicing of theeffector gene to the preceding exon of the target gene; translationframe linker—short DNA sequence ensuring that the effector gene is intranslational frame with the preceding exon of the target gene;self-cleaving peptide—enables separation of the effector protein fromthe target gene product and therefore independent intracellularlocalization for the former; transcript stabilization elements—DNAsequences that facilitate the nuclear export and resulting chimerictranscript stability for enhanced expression (e.g. WPRE sequences, polyAsequences and others). FIG. 3B depicts a Cell-SELECT donor (e.g., donornucleic acid) that preserves the function of the target gene. In someembodiments, this donor nucleic acid can be used to prevent functionaldisruption of the target gene and therefore includes the C-terminalcoding sequence of the targeted gene. In some embodiments, this targetgene-preserving donor nucleic acid prevents functional disruption of thegenomic locus while still affording precise effector gene expression.

The Cell-SELECT donor (e.g., donor nucleic acid) can enable selectiveand high efficiency expression of desired effector genes. The donorconstruct can comprise one or more functional components, including, butnot limited to: a) a recognition site (e.g, a sgRNA cutting site), thatis cleaved in the presence of a programmable nuclease (e.g., Cas9nuclease) and respective targeting molecule (e.g., gRNA) that primes thedonor for genomic integration through NHEJ; b) splice acceptor site,which can comprise both branch point and polypyrimidine stretches thatfunction to capture the splice machinery after transcription of thehybrid transcript; c) translation frame linker—short DNA sequence thatplaces the downstream effector gene in translational frame with thetargeted gene's exon; d) self-cleaving peptide—this DNA stretch encodesthe sequence of a self-cleaving peptide (e.g., P2A, T2A) that releasesthe effector gene from the targeted gene product enabling independentsubcellular targeting of the effector gene product; e) effector genecoding sequence—this can be any protein encoding gene that brings aboutdesired functional alteration in the targeted cell-type; and/or f) oneor more transcript stabilization sequences—these include DNA sequencesthat ensure extended transcript stability following transcription thatmay include WPRE sequences, polyA sequences (e.g. bGH-polyA, hGH-polyAor others) (FIG. 3A). In embodiments of the disclosed methods requiringthe preservation of the targeted gene function, the Cell-SELECT donorcan also include the C-terminal coding sequence of the targeted genethat reconstitutes a fully functional targeted gene product followed bythe effector gene sequence (FIG. 3B). This design can preclude thedisruption of the targeted gene.

Programmable Nucleases and Targeting Molecules

The programmable nuclease can be capable of inducing a double-strandedDNA break. The programmable nuclease can comprise Streptococcus pyogenesCas9 (SpCas9) and/or Staphylococcus aureus Cas9 (SaCas9). Theprogrammable nuclease can comprise Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1,Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5,Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1,Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c,Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, derivatives thereof, or anycombination thereof. The programmable nuclease can comprise a zincfinger nuclease, TAL effector nuclease, meganuclease, MegaTAL, Tev-mTALEN, MegaTev, homing endonuclease, derivatives thereof, or anycombination thereof. The targeting molecule can be capable ofassociating with the programmable nuclease. The targeting molecule cancomprise single strand DNA or single strand RNA. The targeting moleculecan comprise a single guide RNA (sgRNA). The targeting molecule cancomprise a synthetic nucleic acid. There are provided, in someembodiments, programmable gene editing reagents. In some embodiments,the programmable nuclease comprises a nucleic acid guided DNAendonuclease (e.g., Cas9). Some embodiments of the methods andcompositions provided herein employ Streptococcus pyogenes Cas9(SpCas9), Staphylococcus aureus Cas9 (SaCas9), or any other nucleasethat can be guided to cut specific DNA sequences and introducedouble-stranded DNA breaks fulfills the function of both specifying thetargeted genomic region as well as priming the Cell-SELECT donor forNHEJ dependent genomic integration. Some embodiments of the methods andcompositions provided herein employ a targeting molecule (e.g, a gRNA orother nuclease targeting mechanism). The targeting molecule can fulfilltwo functions in the Cell-SELECT method: 1) it can specify the genomicregion (targeted gene intron) for targeted integration of theCell-SELECT donor; and/or 2) it can function to linearize or cut out theCell-SELECT donor sequence priming it for genomic integration. Thenuclease targeting mechanism can also be an integral part of thenuclease as is the case with TALENs or zinc-finger nucleases (ZFNs).

Compositions and Methods of Administration

All cell-SELECT components described herein can be delivered to desiredtissues by a range of viral or non-viral methods as described herein.The selection of a delivery method can depend on the embodiment,including on the specific type of cells and tissues to which theCell-SELECT method is deployed, whether the cells are dividing ornon-dividing, the efficiency of cellular uptake of particular deliveryreagents in a tissue of interest, the desired duration of delivery andmany other factors. One factor in determining the choice of deliverymethod is the achievement of a sufficiently high nuclear concentrationof the Cell-SELECT donor construct for successful genomic integration.

Targeting molecules (e.g., gRNA or other nuclease activity guidingentities) can be introduced to the cell in one of many formulations thatinclude but are not limited to the following examples: a) guide-RNAexpression construct where the genome targeting and donor linearizinggRNA is expressed under the U6 promoter. The DNA that harbors the gRNAexpression cassette can be introduced to the cell by either non-viralmethods (e.g. electroporation, lipid based transfection, etc.), or aspart of a viral vector based delivery strategy (e.g. AAV or lentiviralvectors); b) as a ribonucleoprotein (RNP) complex where the gRNA formsan active complex with the programmable nuclease prior to delivery; c)delivered in the RNA form; and/or d) synthetic non-RNA based guidepolymers. The disclosed Cell-SELECT donors can be delivered in variousformulations depending on the embodiment. For example, they can betargeted to the cell nucleus as DNA plasmids (e.g. in a minicircleplasmid form lacking any superfluous components or regular plasmids withbacterial backbone). They can also be delivered with a viral vector(e.g. AAV or lentiviral vector) that comprises the donor sequences andcan be partially or fully integrated to the genome after cleavage ofrespective gRNA target sites. The programmable nuclease can be deliveredin one of many formulations that include but are not limited to thefollowing examples: a) a programmable nuclease can be delivered as a DNAbased expression construct following a promoter sequence, and this canbe delivered as a mini-circle DNA or regular plasmid or part of a viralgenome (e.g. AAV or lentiviral vector) that facilitates the delivery ofthe DNA to the nucleus; b) a ribonucleoprotein that includes the gRNAspecifying the targeted DNA cutting sequence; c) as an mRNA bringingabout transient expression of the nuclease; and/or d) from a transgenicexpression construct in the organism's genome.

Disclosed herein include compositions. In some embodiments, thecomposition comprises: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and/or (iii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid. Theprogrammable nuclease, the targeting molecule, and/or the donor nucleicacid can be encoded on the same nucleic acid. The programmable nuclease,the targeting molecule, and/or the donor nucleic acid can be encoded ondifferent nucleic acids. In some embodiments, the nucleic acid encodingthe programmable nuclease, the nucleic acid encoding the targetingmolecule, and/or the nucleic acid encoding the donor nucleic acidcomprise DNA and/or RNA. The nucleic acid encoding the programmablenuclease, the nucleic acid encoding the targeting molecule, and/or thenucleic acid encoding the donor nucleic acid can be component(s) of avector. The vector can comprise (i) a programmable nuclease or a nucleicacid encoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and/or (iii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid. Thevector can be an AAV vector, a lentivirus, an integration-deficientlentivirus (IDLV), a plasmid vector, a naked DNA vector, a lipidnanoparticle, or any combination thereof. The plasmid vector cancomprise a minicircle plasmid. The AAV vector can comprise AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or any combination thereof.The vector can be a neurotropic viral vector. The neurotropic viralvector can comprise or can be derived from Herpesviridae, varicellazoster virus, pseudorabies virus, cyromegalovirus, Epstein-barr virus,encephalitis virus, polio virus, coxsackie virus, echo virus, mumpsvirus, measles virus, rabies virus, or any combination thereof. Thevector can comprise a heterologous promoter that drives expression ofthe programmable nuclease, the targeting molecule, and/or the donornucleic acid. The heterologous promoter can be an inducible promoter.The programmable nuclease, the targeting molecule, and/or the donornucleic acid can be encoded by a transgenic construct in the genome ofthe cell. The (i) a programmable nuclease or a nucleic acid encoding theprogrammable nuclease; (ii) a targeting molecule or a nucleic acidencoding the targeting molecule, and/or (iii) a donor nucleic acid or anucleic acid encoding the donor nucleic acid can be a component of aribonucleoprotein (RNP) complex. As disclosed herein, glycoprotein ofthe viral vector can be modified so that the viral vector is targeted toa particular target environment of interest such as central nervoussystem, and to enhance tropism to the target environment of interest(e.g, CNS tropism). In some embodiments, the viral vector delivers apolynucleotide to the heart, peripheral nerves, or a combinationthereof. Pharmaceutical compositions can be prepared, for example, asinjectable formulations.

In some embodiments, the vector can comprise an adenovirus vector, anadeno-associated virus vector, an Epstein-Barr virus vector, a Herpesvirus vector, an attenuated HIV vector, a retroviral vector, a vacciniavirus vector, or any combination thereof. In some embodiments, thevector can comprise an RNA viral vector. In some embodiments, the vectorcan be derived from one or more negative-strand RNA viruses of the orderMononegavirales. In some embodiments, the vector can be a rabies viralvector. Many such vectors useful for transferring exogenous genes intotarget mammalian cells are available. The vectors may be episomal, e.g.plasmids, virus-derived vectors such cytomegalovirus, adenovirus, etc.,or may be integrated into the target cell genome, through homologousrecombination or random integration, e.g. retrovirus-derived vectorssuch as MMLV, HIV-1, ALV, etc. In some embodiments, combinations ofretroviruses and an appropriate packaging cell line may also find use,where the capsid proteins will be functional for infecting the targetcells. Retroviral vectors can be “defective”, i.e. unable to produceviral proteins required for productive infection. Replication of thevector can require growth in the packaging cell line. The term “vector”,as used herein, can refer to a nucleic acid construct designed fordelivery to a host cell or for transfer between different host cells. Asused herein, a vector can be viral or non-viral. The term “vector”encompasses any genetic element that is capable of replication whenassociated with the proper control elements and that can transfer genesequences to cells. A vector can include, but is not limited to, acloning vector, an expression vector, a plasmid, phage, transposon,cosmid, artificial chromosome, virus, virion, etc. There are provided,in some embodiments, expression vectors. As used herein, the term“expression vector” refers to a vector that directs expression of an RNAor polypeptide (e.g., a Cell-SELECT component) from nucleic acidsequences contained therein linked to transcriptional regulatorysequences on the vector. The sequences expressed will often, but notnecessarily, be heterologous to the cell. An expression vector maycomprise additional elements, for example, the expression vector mayhave two replication systems, thus allowing it to be maintained in twoorganisms, for example in human cells for expression and in aprokaryotic host for cloning and amplification. The term “expression”refers to the cellular processes involved in producing RNA and proteinsand as appropriate, secreting proteins, including where applicable, butnot limited to, for example, transcription, transcript processing,translation and protein folding, modification and processing.“Expression products” include RNA transcribed from a gene, andpolypeptides obtained by translation of mRNA transcribed from a gene.The term “gene” means the nucleic acid sequence which is transcribed(DNA) to RNA in vitro or in vivo when operably linked to appropriateregulatory sequences. The gene may or may not include regions precedingand following the coding region, e.g. 5′ untranslated (5′ UTR) or“leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

Integrating vectors have their delivered RNA/DNA permanentlyincorporated into the host cell chromosomes. Non-integrating vectorsremain episomal which means the nucleic acid contained therein is neverintegrated into the host cell chromosomes. Examples of integratingvectors include retroviral vectors, lentiviral vectors, hybridadenoviral vectors, and herpes simplex viral vector. One example of anon-integrative vector is a non-integrative viral vector.Non-integrative viral vectors eliminate the risks posed by integrativeretroviruses, as they do not incorporate their genome into the host DNA.One example is the Epstein Barr oriP/Nuclear Antigen-1 (“EBNA1”) vector,which is capable of limited self-replication and known to function inmammalian cells. As containing two elements from Epstein-Barr virus,oriP and EBNA1, binding of the EBNA1 protein to the virus repliconregion oriP maintains a relatively long-term episomal presence ofplasmids in mammalian cells. This particular feature of the oriP/EBNA1vector makes it ideal for generation of integration-free iPSCs. Anothernon-integrative viral vector is adenoviral vector and theadeno-associated viral (AAV) vector. Other non-integrative viral vectorscontemplated herein are single-strand negative-sense RNA viral vectors,such Sendai viral vector and rabies viral vector. Another example of anon-integrative vector is a minicircle vector. Minicircle vectors arecircularized vectors in which the plasmid backbone has been releasedleaving only the eukaryotic promoter and cDNA(s) that are to beexpressed. As used herein, the term “viral vector” can refer to anucleic acid vector construct that includes at least one element ofviral origin and has the capacity to be packaged into a viral vectorparticle. The viral vector can contain a nucleic acid encoding apolypeptide as described herein in place of nonessential viral genes.The vector and/or particle may be utilized for the purpose oftransferring nucleic acids into cells either in vitro or in vivo.Numerous forms of viral vectors are known in the art.

In some embodiment, the vectors can include a regulatory sequence thatallows, for example, the translation of multiple proteins from a singlemRNA. Non-limiting examples of such regulatory sequences includeinternal ribosome entry site (IRES) and 2A self-processing sequence. Insome embodiments, the 2A sequence is a 2A peptide site fromfoot-and-mouth disease virus (F2A sequence). In some embodiments, theF2A sequence has a standard furin cleavage site. In some embodiments,the vector can also comprise regulatory control elements known to one ofskill in the art to influence the expression of the RNA and/or proteinproducts encoded by the polynucleotide within desired cells of thesubject. In some embodiments, functionally, expression of thepolynucleotide is at least in part controllable by the operably linkedregulatory elements such that the element(s) modulates transcription ofthe polynucleotide, transport, processing and stability of the RNAencoded by the polynucleotide and, as appropriate, translation of thetranscript. A specific example of an expression control element is apromoter, which is usually located 5′ of the transcribed sequence.Another example of an expression control element is an enhancer, whichcan be located 5′ or 3′ of the transcribed sequence, or within thetranscribed sequence. Another example of a regulatory element is arecognition sequence for a microRNA. Another example of a regulatoryelement is an ration and the splice donor and splice acceptor sequencesthat regulate the splicing of said intron. Another example of aregulatory element is a transcription termination signal and/or apolyadenylation sequence.

An effective amount of the disclosed Cell-SELECT reagents can beintroduced into a cell and/or delivered into a subject may any meansknown in the art. The method can comprise: isolating the cell from thesubject prior to the introducing step. The method can comprise:administering the cell into a subject after the introducing step. Theintroducing step can be performed in vivo, in vitro, and/or ex vivo. Theintroducing step can comprise calcium phosphate transfection,DEAE-dextran mediated transfection, cationic lipid-mediatedtransfection, electroporation, electrical nuclear transport, chemicaltransduction, electrotransduction, Lipofectamine-mediated transfection,Effectene-mediated transfection, lipid nanoparticle (LNP)-mediatedtransfection, or any combination thereof. The introducing step cancomprise administering (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and/or (iii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid to asubject comprising the cell. The introducing step can compriseadministering a vector to a subject comprising the cell. The vector cancomprise (i) a programmable nuclease or a nucleic acid encoding theprogrammable nuclease; (ii) a targeting molecule or a nucleic acidencoding the targeting molecule, and/or (iii) a donor nucleic acid or anucleic acid encoding the donor nucleic acid.

The administering can comprise aerosol delivery, nasal delivery, vaginaldelivery, rectal delivery, buccal delivery, ocular delivery, localdelivery, topical delivery, intracisternal delivery, intraperitonealdelivery, oral delivery, intramuscular injection, intravenous injection,subcutaneous injection, intranodal injection, intratumoral injection,intraperitoneal injection, and/or intradermal injection, or anycombination thereof. There are provided, in some embodiments,pharmaceutical composition for administration of any of the compositionsprovided herein. The pharmaceutical composition can be formulated with apharmaceutically acceptable carrier or excipient. A pharmaceuticallyacceptable carrier or excipient refers to a carrier (e.g., carrier,media, diluent, solvent, vehicle, etc.) which does not significantlyinterfere with the biological activity or effectiveness of the activeingredient(s) of a pharmaceutical composition and which is notexcessively toxic to the host at the concentrations at which it is usedor administered. Other pharmaceutically acceptable ingredients can bepresent in the composition as well. Suitable substances and their usefor the formulation of pharmaceutically active compounds are well knownin the art. A pharmaceutical composition is typically formulated to becompatible with its intended route of administration. For topicalapplication, a pharmaceutical composition may be formulated in asuitable ointment, lotion, gel, or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers suitable for use in such compositions. For someapplications, the composition is formulated as a solid (e.g.,lyophilized), liquid, gel, or hydrogel and may contain additives such assurfactants, buffers (e.g., succinate), salts (e.g., sodium chloride),polymers (e.g., polysaccharides, hyaluronic acid), proteins (e.g.,albumin, human serum albumin), or amino acids (e.g., methionine).

Also disclosed herein are pharmaceutical compositions comprising one ormore of the Cell-SELECT reagents disclosed herein and one or morepharmaceutically acceptable carriers. The compositions can also compriseadditional ingredients such as diluents, stabilizers, excipients, andadjuvants. As used herein, “pharmaceutically acceptable” carriers,excipients, diluents, adjuvants, or stabilizers are the ones nontoxic tothe cell or subject being exposed thereto (preferably inert) at thedosages and concentrations employed or that have an acceptable level oftoxicity as determined by the skilled practitioners.

The carriers, diluents and adjuvants can include buffers such asphosphate, citrate, or other organic acids: antioxidants such asascorbic acid; low molecular weight polypeptides (e.g., less than about10 residues); proteins such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine, or lysine;monosaccharides, di saccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics orpolyethylene glycol (PEG). In some embodiments, the physiologicallyacceptable carrier is an aqueous pH buffered solution.

Titers of the disclosed Cell-SELECT reagents to be administered willvary depending, for example, on the particular delivery composition(e.g., viral vector), the mode of administration, the treatment goal,the individual, and the cell type(s) being targeted, and can bedetermined by methods standard in the art.

Actual administration of the disclosed Cell-SELECT reagents can beaccomplished by using any physical method that will transport thedisclosed Cell-SELECT reagents into the cells (e.g., target tissue) ofthe subject. For example, the disclosed Cell-SELECT reagents can beadministered intravenously.

A therapeutically effective amount of the disclosed Cell-SELECT reagentscan be administered to a subject at various points of time. For example,the disclosed Cell-SELECT reagents can be administered to the subjectprior to, during, or after the subject has developed a disease,disorder, and/or infection. The disclosed Cell-SELECT reagents can alsobe administered to the subject prior to, during, or after the occurrenceof a disease, disorder, and/or infection. In some embodiments, thedisclosed Cell-SELECT reagents are administered to the subject duringremission of the disease or disorder. In some embodiments, the disclosedCell-SELECT reagents are administered prior to the onset of the diseaseor disorder in the subject. In some embodiments, the disclosedCell-SELECT reagents are administered to a subject at a risk ofdeveloping the disease or disorder.

Administering can comprise intracranial injection, aerosol delivery,nasal delivery, vaginal delivery, rectal delivery, buccal delivery,ocular delivery, local delivery, topical delivery, intracisternaldelivery, intraperitoneal delivery, oral delivery, intramuscularinjection, intravenous injection, subcutaneous injection, intranodalinjection, intratumoral injection, intraperitoneal injection, and/orintradermal injection, or any combination thereof. Administering cancomprise an injection into a brain region. The brain region can comprisethe Lateral parabrachial nucleus, brainstem, Medulla oblongata,Medullary pyramids, Olivary body, Inferior olivary nucleus, Rostralventrolateral medulla, Respiratory center, Dorsal respiratory group,Ventral respiratory group, Pre-Bötzinger complex, Botzinger complex,Paramedian reticular nucleus, Cuneate nucleus, Gracile nucleus,Intercalated nucleus, Area postrema, Medullary cranial nerve nuclei,Inferior salivatory nucleus, Nucleus ambiguus, Dorsal nucleus of vagusnerve, Hypoglossal nucleus, Solitary nucleus, Pons, Pontine nuclei,Pontine cranial nerve nuclei, chief or pontine nucleus of the trigeminalnerve sensory nucleus (V), Motor nucleus for the trigeminal nerve (V),Abducens nucleus (VI), Facial nerve nucleus (VII), vestibulocochlearnuclei (vestibular nuclei and cochlear nuclei) (VIII), Superiorsalivatory nucleus, Pontine tegmentum, Respiratory centers, Pneumotaxiccenter, Apneustic center, Pontine micturition center (Barrington'snucleus), Locus coeruleus, Pedunculopontine nucleus, Laterodorsaltegmental nucleus, Tegmental pontine reticular nucleus, Superior olivarycomplex, Paramedian pontine reticular formation, Cerebellar peduncles,Superior cerebellar peduncle, Middle cerebellar peduncle, Inferiorcerebellar peduncle, Cerebellum, Cerebellar vermis, Cerebellarhemispheres, Anterior lobe, Posterior lobe, Flocculonodular lobe,Cerebellar nuclei, Fastigial nucleus, Interposed nucleus, Globosenucleus, Emboliform nucleus, Dentate nucleus, Tectum, Corporaquadrigemina, inferior colliculi, superior colliculi, Pretectum,Tegmentum, Periaqueductal gray, Parabrachial area, Medial parabrachialnucleus, Subparabrachial nucleus (Kölliker-Fuse nucleus), Rostralinterstitial nucleus of medial longitudinal fasciculus, Midbrainreticular formation, Dorsal raphe nucleus, Red nucleus, Ventraltegmental area, Substantia nigra, Pars compacta, Pars reticulata,Interpeduncular nucleus, Cerebral peduncle, Crus cerebri, Mesencephaliccranial nerve nuclei, Oculomotor nucleus (III), Trochlear nucleus (IV),Mesencephalic duct (cerebral aqueduct, aqueduct of Sylvius), Pinealbody, Habenular nucleim Stria medullares, Taenia thalami, Subcommissuralorgan, Thalamus, Anterior nuclear group, Anteroventral nucleus (akaventral anterior nucleus), Anterodorsal nucleus, Anteromedial nucleus,Medial nuclear group, Medial dorsal nucleus, Midline nuclear group,Paratenial nucleus, Reuniens nucleus, Rhomboidal nucleus, Intralaminarnuclear group, Centromedial nucleus, Parafascicular nucleus, Paracentralnucleus, Central lateral nucleus, Central medial nucleus, Lateralnuclear group, Lateral dorsal nucleus, Lateral posterior nucleus,Pulvinar, Ventral nuclear group, Ventral anterior nucleus, Ventrallateral nucleus, Ventral posterior nucleus, Ventral posterior lateralnucleus, Ventral posterior medial nucleus, Metathalamus, Medialgeniculate body, Lateral geniculate body, Thalamic reticular nucleus,Hypothalamus, limbic system, HPA axis, preoptic area, Medial preopticnucleus, Suprachiasmatic nucleus, Paraventricular nucleus, Supraopticnucleusm Anterior hypothalamic nucleus, Lateral preoptic nucleus, medianpreoptic nucleus, periventricular preoptic nucleus, Tuberal, Dorsomedialhypothalamic nucleus, Ventromedial nucleus, Arcuate nucleus, Lateralarea, Tuberal part of Lateral nucleus, Lateral tuberal nuclei,Mammillary nuclei, Posterior nucleus, Lateral area, Optic chiasm,Subfornical organ, Periventricular nucleus, Pituitary stalk, Tubercinereum, Tuberal nucleus, Tuberomammillary nucleus, Tuberal region,Mammillary bodies, Mammillary nucleus, Subthalamus, Subthalamic nucleus,Zona incerta, Pituitary gland, neurohypophysis, Pars intermedia,adenohypophysis, cerebral hemispheres, Corona radiata, Internal capsule,External capsule, Extreme capsule, Arcuate fasciculus, Uncinatefasciculus, Perforant Path, Hippocampus, Dentate gyms, Cornu ammonis,Cornu ammonis area 1, Cornu ammonis area 2, Cornu ammonis area 3, Cornuammonis area 4, Amygdala, Central nucleus, Medial nucleus (accessoryolfactory system), Cortical and basomedial nuclei, Lateral andbasolateral nuclei, extended amygdala, Stria terminalis, Bed nucleus ofthe stria terminalis, Claustrum, Basal ganglia, Striatum, Dorsalstriatum (aka neostriatum), Putamen, Caudate nucleus, Ventral striatum,Striatum, Nucleus accumbens, Olfactory tubercle, Globus pallidus,Subthalamic nucleus, Basal forebrain, Anterior perforated substance,Substantia innominata, Nucleus basalis, Diagonal band of Broca, Septalnuclei, Medial septal nuclei, Lamina terminalis, Vascular organ oflamina terminalis, Olfactory bulb, Piriform cortex, Anterior olfactorynucleus, Olfactory tract, Anterior commissure, Uncus, Cerebral cortex,Frontal lobe, Frontal cortex, Primary motor cortex, Supplementary motorcortex, Premotor cortex, Prefrontal cortex, frontopolar cortex,Orbitofrontal cortex, Dorsolateral prefrontal cortex, dorsomedialprefrontal cortex, ventrolateral prefrontal cortex, Superior frontalgyms, Middle frontal gyms, Inferior frontal gyms, Brodmann areas (4, 6,8, 9, 10, 11, 12, 24, 25, 32, 33, 44, 45, 46, and/or 47), Parietal lobe,Parietal cortex, Primary somatosensory cortex (S1), Secondarysomatosensory cortex (S2), Posterior parietal cortex, postcentral gyms,precuneus, Brodmann areas (1, 2, 3 (Primary somesthetic area), 5, 7, 23,26, 29, 31, 39, and/or 40), Occipital lobe, Primary visual cortex (V1),V2, V3, V4, V5/MT, Lateral occipital gyms, Cuneus, Brodmann areas (17(V1, primary visual cortex), 18, and/or 19), temporal lobe, Primaryauditory cortex (A1), secondary auditory cortex (A2), Inferior temporalcortex, Posterior inferior temporal cortex, Superior temporal gyms,Middle temporal gyms, Inferior temporal gyms, Entorhinal Cortex,Perirhinal Cortex, Parahippocampal gyms, Fusiform gyms, Brodmann areas(9, 20, 21, 22, 27, 34, 35, 36, 37, 38, 41, and/or 42), Medial superiortemporal area (MST), insular cortex, cingulate cortex, Anteriorcingulate, Posterior cingulate, dorsal cingulate, Retrosplenial cortex,Indusium griseum, Subgenual area 25, Brodmann areas (23, 24; 26, 29, 30(retrosplenial areas), 31, and/or 32), cranial nerves (Olfactory (I),Optic (II), Oculomotor (III), Trochlear (IV), Trigeminal (V), Abducens(VI), Facial (VII), Vestibulocochlear (VIII), Glossopharyngeal (IX),Vagus (X), Accessory (XI), Hypoglossal (XII)), or any combinationthereof.

The brain region can comprise neural pathways Superior longitudinalfasciculus, Arcuate fasciculus, Thalamocortical radiations, Cerebralpeduncle, Corpus callosum, Posterior commissure, Pyramidal orcorticospinal tract, Medial longitudinal fasciculus, dopamine system,Mesocortical pathway, Mesolimbic pathway, Nigrostriatal pathway,Tuberoinfundibular pathway, serotonin system, Norepinephrine Pathways,Posterior column-medial lemniscus pathway, Spinothalamic tract, Lateralspinothalamic tract, Anterior spinothalamic tract, or any combinationthereof.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure.

Example 1 Targeted Expression of an Effector Gene in Molecularly DefinedCell Types in Cell Culture by the Cell-SELECT Method

This example provides proof of concept for the Cell-SELECT methodprovided herein. It was demonstrated that the Cell-SELECT constructsdescribed herein can reliably and robustly reproduce the expressionpattern of the genetic locus being targeted. This was initially done inprimary neural cultures. The gene editing machinery (e.g., guide RNA)was designed to target Cell-SELECT construct integration to the firstintron of a pan-neuronally expressed gene (Tubb3). The Cell-SELECT donorvectors carried a fluorescent reporter gene for rapid integrationread-out. In some embodiments, intronic integration, splice acceptor andtranscript stabilization components were optimized to yield the highestefficiency expression of Cell-SELECT delivered genes. Some embodimentsof the Cell-SELECT donor and guide RNA combinations were furthervalidated in vivo by adeno-associated virus dependent vector deliverythrough stereotactic brain injections (see below Examples). FIGS. 4A-4Edemonstrate the ability of the disclosed Cell-SELECT method to targetthe expression of the effector gene to a molecularly defined cell typesin cell culture. FIG. 4A shows that the gene Tubb3 was expressed in theneuroblastoma cell line N2A whereas it was absent in the fibroblast cellline NIH-3T3 (scale bar—25 microns). Tubb3 IHC—immunohistochemicallabeling of Tubb3 protein. DAPI—nuclear stain. FIG. 4B depicts anexemplary experimental design for demonstrating cell-type-specifictargeting efficiency of the Cell-SELECT technology. The cell-SELECTdonor was knocked into the Tubb3 gene locus. Since Tubb3 was onlyexpressed in one of the tested cell lines (N2A) and not expressed in theother (NIH-3T3), the effector gene was expected to be active (cellslabeled by GFP fluorescence) only in Tubb3 expressing cells. TheCell-SELECT effector gene was spliced to the exon of thecell-type-specifically expressed gene (Tubb3) and during translationreleased as an independent protein due to self-cleavage of the P2Apeptide linker. SA—splice acceptor. P2A—self cleaving linker peptidesequence, GFP—green fluorescent protein. In some embodiments, theCell-SELECT donor construct comprises SEQ ID NO: 1. FIG. 4C shows thattransfection of cells lacking the expression of the targeted Tubb3 gene(NIH-3T3) with Cell-SELECT knock-in construct carrying the GFP effectorgene, Cas9 nuclease and Tubb3 intron targeting gRNA does not lead toexpression of the effector gene. The control condition lacks Cas9nuclease, and the test condition includes all three Cell-SELECT geneediting components. gRNA expression cassette includes the red mCherryreporter, and successful effector gene activity labels cells green. FIG.4D shows that transfection of Tubb3 expressing cells (N2A) gene withCell-SELECT knock-in construct carrying the GFP effector gene, Cas9nuclease and Tubb3 intron targeting gRNA leads to high efficiencyexpression of the effector gene (GFP). Overlap between gRNA andsuccessful effector-gene expressing cells shown in yellow in the“Overlay” panel. The control condition lacks Cas9 nuclease, and the testcondition includes all three Cell-SELECT gene editing components. FIG.4E shows the quantification of cell-type-specific targeting efficiencyof the Cell-SELECT technology (n=8, ***−p<0.001, unpaired t-test).

Example 2 Comparison of HITI-CRISPR and Cell-SELECT Methods in CellCulture and Live Animals

This example provides proof of concept for the Cell-SELECT methodprovided herein and demonstrates the several fold higher cell targetingefficiencies achieved with the disclosed methods and compositions ascompared to currently available solutions. FIGS. 5A-5C demonstrate thehigh yield knock-in efficiency of the Cell-SELECT method as compared toother gene editing based gene tagging technologies in a cell culturemodel expressing the targeted Tubb3 gene (N2A cell line). FIG. 5A showsthe regular gene tagging approach that useshomology-independent-targeted integration (HITI) CRISPR based knock-inof the GFP reporter at the stop-codon of the Tubb3 gene. The sensitivityof the traditional gene tagging methods to imprecisions of the NHEJdependent DNA repair and of Cas9 cleavage result in modest effector geneexpression in cells where the target gene was expressed. The controlcondition lacks Cas9 expression. The test condition cells have beentransfected with Cas9, Tubb3 stop codon targeting gRNA and promoterlessGFP knock-in construct. SEQ ID NO: 2 comprises a pAAV-CELL-SELECTconstruct for targeted viral knock-in of fluorescent reporter EYFP. Insome embodiments, the expression of the targeting molecule (e.g., gRNA)can be driven by an RNA polymerase III promoter (e.g., U6 snRNApromoter). FIG. 5B shows the high efficiency of Cell-SELECT basedeffector gene (GFP) targeting to Tubb3 expressing cells. Cell-SELECTdonor insertions was guided to the first intron of the Tubb3 gene andthe effector gene was spliced to the expressed Tubb3 first exon.Effector gene expression was dependent on the presence of Cas9 nuclease.FIG. 5C shows the quantification of cell targeting efficiencies withregular HITI-CRISPR based gene tagging and contrasts that to theCell-SELECT method. Cell-SELECT achieves 2.5× higher cellular targetingefficiency as compared to the state of the art gene editing basedeffector-gene knock-in approaches (n=8, ***−p<0.001, unpaired t-test).FIGS. 6A-6C shows that Cell-SELECT based in vivo gene editing asdisclosed herein achieve efficient and high levels of effector geneexpression in desired cell types in live animals. FIG. 6A shows anon-limiting exemplary schematic illustration of the injection andtesting of AAV viral vector based in vivo gene editing in the mousebrain. Effector gene (GFP) carrying HITI CRISPR or Cell-SELECT templatesand gRNA targeting effector gene integration to the pan-neuronallyexpressed Tubb3 locus were employed. Cas9 gene was supplied in transfrom H11 locus. AAV injections (300 nL of virus at 10{circumflex over( )}12 virus particles/ml) were directed to the CA1 region ofhippocampus. Cell-SELECT was found to achieve efficient and high levelsof effector gene expression in desired cell types in vivo reaching morethan an order of magnitude higher effector gene knock-in efficienciesthan regular HITI-CRISPR based gene tagging. FIG. 6B depicts Tubb3integration site and representative knock in efficiency with regularHITI-CRISPR based in vivo gene editing. FIG. 6C depicts Tubb3integration site and representative knock in efficiency with Cell-SELECTbased in vivo gene editing.

Example 3 In Vivo Gene Editing

FIGS. 7A-7B demonstrate the cell targeting efficiency of Tubb3expressing neurons with in vivo gene editing based effector geneknock-in. FIG. 7A shows a non-limiting exemplary schematic illustrationof the experimental design, where AAV-based viral vectors were used todeliver the knock-in construct carrying the GFP gene and gene editingnuclease reagents to the mouse hippocampal CA1 region via stereotacticinjections. FIG. 7B shows the results of an experiment for anunoptimized targeted in vivo genomic integration of a GFP construct intothe pan-neuronally expressed Tubb3 locus using HITI CRISPR basedknock-in donor introduction. The experiment yielded ˜10% targetingefficiency for the effector gene expression with successful targetingtaking place only in the presence of the Cas9 recombinase.

Example 4 Identification of a Candidate Locus for Cell-SELECT Targeting

This example provides further validation for the Cell-SELECT methoddisclosed herein. In some embodiments, the methods and compositionsprovided herein can be employed to manipulate behavior-mediating neurontypes in a mammalian brain. The disclosed Cell-SELECT methods can beused as a method to functionally manipulate molecularly defined neuronsin the mammalian central nervous system that control essentialphysiological or behavioral functions. For example, the expression ofpain is mediated by activity in molecularly distinct cell types invarious mammalian brain centers such as the lateral parabrachial nucleus(LPBN). These cell types differ from other neurons in these braincenters by virtue of distinct gene expression programs that are activein these cells.

FIGS. 8A-8B show the neural activity induced immediate early geneexpression in control condition or after pain induction in molecularcell-types in the major mammalian brain pain relay—lateral parabrachialnucleus (LPBN). Single cell RNA-seq in the midbrain nucleus LPBNrevealed two cell types that are robustly activated by exposure to pain(Glut1, Glut2, red arrows). Width and height of violin plots indicatesproportion of cells expressing a given gene. FIG. 8C shows theexpression of pain cell type labeling molecular marker gene Nr4a1. Painactivated cells are selectively labeled by the expression of Nr4a1 geneidentifying candidate locus for Cell-SELECT targeting. Thus, anidentification of two pain activated molecular cell types in the mousemidbrain nucleus LPBN—a pain relay in the central nervous system that isessential for pain perception was made as shown in FIGS. 8A-8C. Thiscontext is an ideal challenge, as the LPBN contains 14 other molecularcell types that regulate taste, blood pressure, breathing and otheressential physiological functions. Cells that are essential for thedetection of pain and are concomitantly driven by peripheral noxiousstimuli express the gene Nr4a1 (FIGS. 8A-8C). The Cell-SELECT methodsprovided herein can be used to control the animal's sensitivity to paininducing stimuli by delivering a synaptic function blocking gene TNT toLPBN neurons with AAV viral vectors carrying the Cell-SELECT knock indonor as well as Cas9 and gRNA constructs targeting Cell-SELECT donorintegration to the first intron of Nr4a1 gene. This can result inselective synaptic blockade of pain processing neurons in LPBN andconsequently downregulates the pain experience by directly functionallyre-engineering the underlying circuit function.

Single cell profiling by RNA-seq revealed both molecular cell types aswell as cell type specifically expressed genetic programs. The disclosedsingle cell RNA-seq data from LPBN has identified several genes that areselectively expressed in pain neurons (e.g. Nr4a1 in FIG. 8C). Thisinformation enables the design of an AAV viral vector deliveringCell-SELECT constructs containing an effector gene (e.g., fluorescentreporter) targeting the intron of one or more of those genes. In someembodiments, Cell-SELECT reagents can be delivered to the midbrain bystereotactic brain injection. The analysis provided herein identifiesgenetic loci that reliably and selectively label pain mediatingmolecular neuron types in the midbrain. In some embodiments, theeffector gene comprises a synaptic or chronic activity blocking gene(e.g., TNT, Kir2.1 etc.) to functionally silence pain mediating cells inLPBN, which can result in changes in pain sensitivity and perception ina panel of acute and chronic pain assays.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise. Any reference to “or” herein isintended to encompass “and/or” unless otherwise stated.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1. A method of incorporating an effector gene into the genome of a cell,comprising: introducing into a cell: (i) a programmable nuclease or anucleic acid encoding the programmable nuclease; (ii) a targetingmolecule or a nucleic acid encoding the targeting molecule, and (iii) adonor nucleic acid or a nucleic acid encoding the donor nucleic acid,wherein the donor nucleic acid comprises a recognition site, a spliceacceptor site, a self-cleaving peptide sequence, an effector gene, andan optional transcript stabilization element, wherein the cell comprisesa target gene differentially expressed in a unique cell type and/or in acell during a unique cell state, wherein the target gene comprises anintron comprising the recognition site, and wherein the targetingmolecule is complementary to the recognition site and the programmablenuclease is capable of cleaving the recognition site, whereby the donornucleic acid is capable of being incorporated into the intron throughnon-homologous end joining (NHEJ)-dependent DNA repair.
 2. (canceled) 3.(canceled)
 4. (canceled)
 5. The method of claim 1, wherein the donornucleic acid comprises a translation frame linker, optionally whereinthe translation frame linker places the effector gene in translationalframe with the preceding exon of the target gene, further optionallywherein the donor nucleic acid comprises the structure 5′-[recognitionsite]-[splice acceptor site]-[translation frame linker]-[self-cleavingpeptide sequence]-[effector gene]-3′.
 6. The method of claim 1, whereinthe donor nucleic acid comprises the target gene coding sequencedownstream of the intron, optionally wherein the donor nucleic acidcomprises the structure 5′-[recognition site]-[splice acceptorsite]-[target gene coding sequence downstream of theintron]-[self-cleaving peptide sequence]-[effector gene]-3′.
 7. Themethod of claim 1, wherein the recognition site of the donor nucleicacid and the recognition site of the intron are the same, optionallywherein the recognition site does not exist after the donor nucleic acidhas been properly incorporated into the intron, further optionallywherein the recognition site is adjacent to a protospacer adjacent motif(PAM) capable of being recognized by the programmable nuclease. 8.(canceled)
 9. The method of claim 1, wherein the expression of theeffector gene in a cell other than the unique cell type is less thanabout 5 percent of the expression of the effector gene in the uniquecell type.
 10. (canceled)
 11. The method of claim 1, wherein theexpression of the effector gene in a cell not in the unique cell stateis less than about 10 percent of the expression of the effector gene ina cell during the unique cell state.
 12. The method of claim 1, whereinthe effector gene is only expressed in a cell expressing the targetgene.
 13. The method of claim 1, wherein the expression of the effectorgene in a cell that does not express the target gene is less than about5 percent of the expression of the effector gene in a cell that doesexpress the target gene.
 14. (canceled)
 15. (canceled)
 16. (canceled)17. (canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. Themethod of claim 1, wherein the unique cell type and/or the cell in theunique cell state is associated with the pathology of a disease ordisorder.
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)26. The method of claim 1, wherein the expression of the effector geneis capable of modulating cellular membrane potential, optionally whereinthe expression of the effector gene is capable of altering the membranepotential of the cell by depolarizing the cell and/or hyperpolarizingthe cell.
 27. The method of claim 1, wherein the expression of theeffector gene is capable of reducing synaptic transmission by at least10 percent.
 28. (canceled)
 29. The method of claim 1, wherein theexpression of the effector gene is capable of sensitizing the cell to adrug, a prodrug, a pharmacological compound, temperature change, orlight.
 30. (canceled)
 31. (canceled)
 32. (canceled)
 33. The method ofclaim 1, wherein the introducing step achieves an at least 10 percenthigher targeting efficiency as compared to a homology directed repair(HDR)-based method.
 34. The method of claim 1, wherein the introducingstep achieves an at least 10 percent higher targeting efficiency ascompared to a Homology Independent Targeted Integration (HITI)-basedmethod integrating an effector gene into an exon.
 35. (canceled) 36.(canceled)
 37. (canceled)
 38. The method of claim 1, wherein the spliceacceptor site is recognizable and cleavable by a spliceosome, optionallywherein the splice acceptor site comprises a branchpoint, apolypyrimidine tract, a 3′ splice site, or any combination thereof. 39.The method of claim 1, wherein the targeting molecule is capable ofassociating with the programmable nuclease, optionally wherein thetargeting molecule comprises single strand DNA or single strand RNA,further optionally wherein the targeting molecule comprises a singleguide RNA (sgRNA).
 40. The method of claim 1, wherein at least two ofthe programmable nuclease, the targeting molecule, and the donor nucleicacid are encoded on the same nucleic acid.
 41. The method of claim 1,wherein at least two of the programmable nuclease, the targetingmolecule, and the donor nucleic acid are encoded on different nucleicacids.
 42. (canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled) 46.The method of claim 1, comprising isolating the cell from a subjectprior to the introducing step.
 47. (canceled)
 48. (canceled)
 49. Themethod of claim 1, wherein the introducing step comprises administering(i), (ii), and/or (iii) to a subject comprising the cell.
 50. The methodof claim 1, wherein the introducing step comprises administering avector to a subject comprising the cell, wherein the vector comprises(i), (ii), and/or (iii).
 51. (canceled)
 52. (canceled)
 53. Acomposition, comprising: (i) a programmable nuclease or a nucleic acidencoding the programmable nuclease; (ii) a targeting molecule or anucleic acid encoding the targeting molecule, and/or (iii) a donornucleic acid or a nucleic acid encoding the donor nucleic acid, whereinthe donor nucleic acid comprises a recognition site, a splice acceptorsite, a self-cleaving peptide sequence, an effector gene, and anoptional transcript stabilization element, and wherein the targetingmolecule is complementary to the recognition site. 54.-97. (canceled)